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author:("shirisha, A")
1.  Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma 
Nature Communications  2015;6:7557.
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Here we studied 60 RMSs using whole-exome/-transcriptome sequencing, copy number (CN) and DNA methylome analyses to unravel the genetic/epigenetic basis of RMS. On the basis of methylation patterns, RMS is clustered into four distinct subtypes, which exhibits remarkable correlation with mutation/CN profiles, histological phenotypes and clinical behaviours. A1 and A2 subtypes, especially A1, largely correspond to alveolar histology with frequent PAX3/7 fusions and alterations in cell cycle regulators. In contrast, mostly showing embryonal histology, both E1 and E2 subtypes are characterized by high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation and in E2, also by p53 inactivation. Despite the better prognosis of embryonal RMS, patients in the E2 are likely to have a poor prognosis. Our results highlight the close relationships of the methylation status and gene mutations with the biological behaviour in RMS.
Rhabdomyosarcoma is a common childhood soft-tissue cancer. Here Seki and Nishimura analyse the exome, transcriptome, copy number and DNA methylome of 60 sarcomas and identify distinct methylation subgroups associated with genetic and clinical features.
PMCID: PMC4506514  PMID: 26138366
2.  Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome 
Nature communications  2015;6:6042.
Somatic mutations in the spliceosome gene ZRSR2 — located on the X chromosome — are associated with myelodysplastic syndrome (MDS). ZRSR2 is involved in the recognition of 3΄ splice site during the early stages of spliceosome assembly; however, its precise role in RNA splicing has remained unclear. Here, we characterize ZRSR2 as an essential component of the minor spliceosome (U12-dependent) assembly. shRNA mediated knockdown of ZRSR2 leads to impaired splicing of the U12-type introns, and RNA-Sequencing of MDS bone marrow reveals that loss of ZRSR2 activity causes increased mis-splicing. These splicing defects involve retention of the U12-type introns while splicing of the U2-type introns remain mostly unaffected. ZRSR2 deficient cells also exhibit reduced proliferation potential and distinct alterations in myeloid and erythroid differentiation in vitro. These data identify a specific role for ZRSR2 in RNA splicing and highlight dysregulated splicing of U12-type introns as a characteristic feature of ZRSR2 mutations in MDS.
PMCID: PMC4349895  PMID: 25586593
3.  A Novel Multivariate Index for Pancreatic Cancer Detection Based On the Plasma Free Amino Acid Profile 
PLoS ONE  2015;10(7):e0132223.
The incidence of pancreatic cancer (PC) continues to increase in the world, while most patients are diagnosed with advanced stages and survive <12 months. This poor prognosis is attributable to difficulty of early detection. Here we developed and evaluated a multivariate index composed of plasma free amino acids (PFAAs) for early detection of PC.
We conducted a cross-sectional study in multi-institutions in Japan. Fasting plasma samples from PC patients (n = 360), chronic pancreatitis (CP) patients (n = 28), and healthy control (HC) subjects (n = 8372) without apparent cancers who were undergoing comprehensive medical examinations were collected. Concentrations of 19 PFAAs were measured by liquid chromatography–mass spectrometry. We generated an index consisting of the following six PFAAs: serine, asparagine, isoleucine, alanine, histidine, and tryptophan as variables for discrimination in a training set (120 PC and matching 600 HC) and evaluation in a validation set (240 PC, 28 CP, and 7772 HC).
Several amino acid concentrations in plasma were significantly altered in PC. Plasma tryptophan and histidine concentrations in PC were particularly low, while serine was particularly higher than that of HC. The area under curve (AUC) based on receiver operating characteristic (ROC) curve analysis of the resulting index to discriminate PC from HC were 0.89 [95% confidence interval (CI), 0.86–0.93] in the training set. In the validation set, AUCs based on ROC curve analysis of the PFAA index were 0.86 (95% CI, 0.84–0.89) for all PC patients versus HC subjects, 0.81 (95% CI, 0.75–0.86) for PC patients from stage IIA to IIB versus HC subjects, and 0.87 (95% CI, 0.80–0.93) for all PC patients versus CP patients.
These findings suggest that the PFAA profile of PC was significantly different from that of HC. The PFAA index is a promising biomarker for screening and diagnosis of PC.
PMCID: PMC4489861  PMID: 26133769
4.  Effects of Anti-G and Anti-F Antibodies on Airway Function after Respiratory Syncytial Virus Infection 
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illnesses in infants worldwide. Both RSV-G and RSV-F glycoproteins play pathogenic roles during infection with RSV. The objective of this study was to compare the effects of anti–RSV-G and anti–RSV-F monoclonal antibodies (mAbs) on airway hyperresponsiveness (AHR) and inflammation after primary or secondary RSV infection in mice. In the primary infection model, mice were infected with RSV at 6 weeks of age. Anti–RSV-G or anti–RSV-F mAbs were administered 24 hours before infection or Day +2 postinfection. In a secondary infection model, mice were infected (primary) with RSV at 1 week (neonate) and reinfected (secondary) 5 weeks later. Anti–RSV-G and anti–RSV-F mAbs were administered 24 hours before the primary infection. Both mAbs had comparable effects in preventing airway responses after primary RSV infection. When given 2 days after infection, anti–RSV-G–treated mice showed significantly decreased AHR and airway inflammation, which persisted in anti–RSV-F–treated mice. In the reinfection model, anti–RSV-G but not anti–RSV-F administered during primary RSV infection in neonates resulted in decreased AHR, eosinophilia, and IL-13 but increased levels of IFN-γ in bronchoalveolar lavage on reinfection. These results support the use of anti–RSV-G in the prevention and treatment of RSV-induced disease.
PMCID: PMC4091856  PMID: 24521403
airway; inflammation; respiratory syncytial virus; anti–respiratory syncytial virus–G; anti–respiratory syncytial virus–F
5.  Action Mechanism of Fibroblast Growth Factor-2 (FGF-2) in the Promotion of Periodontal Regeneration in Beagle Dogs 
PLoS ONE  2015;10(6):e0131870.
Fibroblast growth factor-2 (FGF-2) enhances the formation of new alveolar bone, cementum, and periodontal ligament (PDL) in periodontal defect models. However, the mechanism through which FGF-2 acts in periodontal regeneration in vivo has not been fully clarified yet. To reveal the action mechanism, the formation of regenerated tissue and gene expression at the early phase were analyzed in a beagle dog 3-wall periodontal defect model. FGF-2 (0.3%) or the vehicle (hydroxypropyl cellulose) only were topically applied to the defect in FGF-2 and control groups, respectively. Then, the amount of regenerated tissues and the number of proliferating cells at 3, 7, 14, and 28 days and the number of blood vessels at 7 days were quantitated histologically. Additionally, the expression of osteogenic genes in the regenerated tissue was evaluated by real-time PCR at 7 and 14 days. Compared with the control, cell proliferation around the existing bone and PDL, connective tissue formation on the root surface, and new bone formation in the defect at 7 days were significantly promoted by FGF-2. Additionally, the number of blood vessels at 7 days was increased by FGF-2 treatment. At 28 days, new cementum and PDL were extended by FGF-2. Moreover, FGF-2 increased the expression of bone morphogenetic protein 2 (BMP-2) and osteoblast differentiation markers (osterix, alkaline phosphatase, and osteocalcin) in the regenerated tissue. We revealed the facilitatory mechanisms of FGF-2 in periodontal regeneration in vivo. First, the proliferation of fibroblastic cells derived from bone marrow and PDL was accelerated and enhanced by FGF-2. Second, angiogenesis was enhanced by FGF-2 treatment. Finally, osteoblastic differentiation and bone formation, at least in part due to BMP-2 production, were rapidly induced by FGF-2. Therefore, these multifaceted effects of FGF-2 promote new tissue formation at the early regeneration phase, leading to enhanced formation of new bone, cementum, and PDL.
PMCID: PMC4488280  PMID: 26120833
6.  Entrainment of the mouse circadian clock by sub-acute physical and psychological stress 
Scientific Reports  2015;5:11417.
The effects of acute stress on the peripheral circadian system are not well understood in vivo. Here, we show that sub-acute stress caused by restraint or social defeat potently altered clock gene expression in the peripheral tissues of mice. In these peripheral tissues, as well as the hippocampus and cortex, stressful stimuli induced time-of-day-dependent phase-advances or -delays in rhythmic clock gene expression patterns; however, such changes were not observed in the suprachiasmatic nucleus, i.e. the central circadian clock. Moreover, several days of stress exposure at the beginning of the light period abolished circadian oscillations and caused internal desynchronisation of peripheral clocks. Stress-induced changes in circadian rhythmicity showed habituation and disappeared with long-term exposure to repeated stress. These findings suggest that sub-acute physical/psychological stress potently entrains peripheral clocks and causes transient dysregulation of circadian clocks in vivo.
PMCID: PMC4466793  PMID: 26073568
7.  Light-Induced Conformational Changes in Full-Length Arabidopsis thaliana Cryptochrome 
Journal of molecular biology  2011;413(1):128-137.
Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This interdomain modulation would be critical for CRYs’ ability to transduce a bluelight signal into altered protein–protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.
PMCID: PMC4451184  PMID: 21875594
blue-light receptor; flavin; photoreduction; diffusion; conformational dynamics
8.  Impact of the duration of hormonal therapy following radiotherapy for localized prostate cancer 
Oncology Letters  2015;10(1):255-259.
External-beam radiotherapy (EBRT) combined with androgen deprivation therapy (ADT) is known to provide improved survival outcomes compared with EBRT alone in the treatment of prostate cancer; however, the use of ADT has been reported to be associated with adverse events. Accordingly, the aim of the present study was to clarify the adequate duration of ADT when combined with EBRT to treat patients with high-risk localized prostate cancer, with consideration of survival outcomes and toxicity. Between 2001 and 2011, 173 patients with high-risk localized prostate cancer received ADT combined with EBRT, at a median dose of 69.6 Gy. Of these, 54 (31%) underwent short-term ADT (<36 months) and 119 (69%) underwent long-term ADT (≥36 months). During the median follow-up period of 54 months, the five-year progression-free survival rate of patients receiving short-term ADT (72.9%) was significantly lower than that of patients receiving long-term ADT (92.8%) (P<0.01). Furthermore, the incidence of cardiovascular toxicity at grade II or above was significantly higher amongst patients treated with short-term ADT compared with patients treated with long-term ADT (P<0.01). Thus, the present study determined that ADT for ≥36 months combined with EBRT significantly improved the progression-free survival of patients with high-risk localized prostate cancer and exhibited an acceptable toxicity profile.
PMCID: PMC4487142  PMID: 26171009
androgen deprivation therapy; prostate cancer; external-beam radiotherapy
9.  Three-dimensional morphology of the human embryonic brain 
Data in Brief  2015;4:116-118.
The morphogenesis of the cerebral vesicles and ventricles was visualized in 3D movies using images derived from human embryo specimens between Carnegie stage 13 and 23 from the Kyoto Collection. These images were acquired with a magnetic resonance microscope equipped with a 2.35-T superconducting magnet. Three-dimensional images using the same scale demonstrated brain development and growth effectively. The non-uniform thickness of the brain tissue, which may indicate brain differentiation, was visualized with thickness-based surface color mapping. A closer view was obtained of the unique and complicated differentiation of the rhombencephalon, especially with regard to the internal view and thickening of the brain tissue. The present data contribute to a better understanding of brain and cerebral ventricle development.
PMCID: PMC4510431  PMID: 26217773
10.  Human CLP1 mutations alter tRNA biogenesis affecting both peripheral and central nervous system function 
Cell  2014;157(3):636-650.
CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a novel neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.
PMCID: PMC4146440  PMID: 24766809
11.  Expression of Cancer/Testis Antigens in Prostate Cancer is Associated With Disease Progression 
The Prostate  2010;70(16):1778-1787.
The cancer/testis antigens (CTAs) are a unique group of proteins normally expressed in germ cells but aberrantly expressed in several types of cancers including prostate cancer (PCa). However, their role in PCa has not been fully explored.
CTA expression profiling in PCa samples and cell lines was done utilizing a custom microarray that contained probes for two-thirds of all CTAs. The data were validated by quantitative PCR (Q-PCR). Functional studies were carried out by silencing gene expression with siRNA. DNA methylation was determined by methylation-specific PCR.
A majority of CTAs expressed in PCa are located on the X chromosome (CT-X antigens). Several CT-X antigens from the MAGEA/CSAG subfamilies are coordinately upregulated in castrate-resistant prostate cancer (CRPC) but not in primary PCa. In contrast, PAGE4 is highly upregulated in primary PCa but is virtually silent in CRPC. Further, there was good correlation between the extent of promoter DNA methylation and CTA expression. Finally, silencing the expression of MAGEA2 the most highly upregulated member, significantly impaired proliferation of prostate cancer cells while increasing their chemosensitivity.
Considered together, the remarkable stage-specific expression patterns of the CT-X antigens strongly suggests that these CTAs may serve as unique biomarkers that could potentially be used to distinguish men with aggressive disease who need treatment from men with indolent disease not requiring immediate intervention. The data also suggest that the CT-X antigens may be novel therapeutic targets for CRPC for which there are currently no effective therapeutics.
PMCID: PMC4403006  PMID: 20583133
cancer/testis antigens; prostate cancer; castrate-resistant prostate cancer; biomarker
12.  Antibodies to myelin oligodendrocyte glycoprotein in idiopathic optic neuritis 
BMJ Open  2015;5(4):e007766.
To investigate the differences of clinical features, cerebrospinal fluid (CSF), MRI findings and response to steroid therapies between patients with optic neuritis (ON) who have myelin oligodendrocyte glycoprotein (MOG) antibodies and those who have seronegative ON.
We recruited participants in the department of neurology and ophthalmology in our hospital in Japan.
We retrospectively evaluated the clinical features and response to steroid therapies of patients with ON. Sera from patients were tested for antibodies to MOG and aquaporin-4 (AQP4) with a cell-based assay.
Between April 2009 and March 2014, we enrolled serial 57 patients with ON (27 males, 30 females; age range 16–84 years) who ophthalmologists had diagnosed as having or suspected to have ON with acute visual impairment and declined critical flicker frequency, abnormal findings of brain MRI, optical coherence tomography and fluorescein fundus angiography at their onset or recurrence. We excluded those patients who fulfilled the diagnostic criteria of neuromyelitis optica (NMO)/NMO spectrum disorders (NMOSD), MS McDonald's criteria, and so on. Finally we defined 29 patients with idiopathic ON (14 males, 15 females, age range 16–84 years).
27.6% (8/29) were positive for MOG antibodies and 3.4% (1/29) were positive for AQP4. Among the eight patients with MOG antibodies, five had optic pain (p=0.001) and three had prodromal infection (p=0.179). Three of the eight MOG-positive patients showed significantly high CSF levels of myelin basic protein (p=0.021) and none were positive for oligoclonal band in CSF. On MRIs, seven MOG-positive patients showed high signal intensity on optic nerve, three had a cerebral lesion and one had a spinal cord lesion. Seven of the eight MOG-positive patients had a good response to steroid therapy.
Although not proving primary pathogenicity of anti-MOG antibodies, the present results indicate that the measurement of MOG antibodies is useful in diagnosing and treating ON.
PMCID: PMC4390681  PMID: 25838512
13.  Predictors of post-infectious chronic fatigue syndrome in adolescents 
This study focused on identifying risk factors for adolescent post-infectious chronic fatigue syndrome (CFS), utilizing a prospective, nested case–control longitudinal design in which over 300 teenagers with infectious mononucleosis (IM) were identified through primary care sites and followed. Baseline variables that were gathered several months following IM, included autonomic symptoms, days in bed since IM, perceived stress, stressful life events, family stress, difficulty functioning and attending school, family stress, and psychiatric disorders. A number of variables were predictors of post-infectious CFS at six months; however, when autonomic symptoms were used as a control variable, only days spent in bed since mono was a significant predictor. Step-wise logistic regression findings indicated that baseline autonomic symptoms as well as days spent in bed since mono, which reflect the severity of illness, were the only significant predictors of those who met CFS criteria at six months.
PMCID: PMC3956649  PMID: 24660116
mononucleosis; chronic fatigue syndrome; risk factors; autonomic symptoms; longitudinal
14.  Transcatheter Arterial Coil Embolization of Ruptured Common Hepatic Artery Aneurysm in a Patient with Behçet's Disease 
Case Reports in Radiology  2015;2015:790175.
Hepatic artery aneurysm is a rare and potentially life-threatening entity. We report a case of ruptured common hepatic artery aneurysm in a patient with Behçet's disease. The ruptured aneurysm was treated successfully with transcatheter arterial coil embolization. Transcatheter arterial embolization is the preferred treatment modality in patients at high risk of surgical intervention.
PMCID: PMC4364124  PMID: 25821623
15.  Health anxiety and healthcare costs in Japanese individuals: an Internet survey 
Aim: Health anxiety, also known as hypochondriasis, is classifiable as an anxiety disorder. The aim of this study was to examine the relationship between health anxiety and healthcare costs. Method: Participants – 100 Japanese individuals from the general population with chronic health problems and 100 without chronic health problems – were recruited via the Internet. They completed self-report scales measuring health anxiety, state anxiety, depression, obsessionality, and a scale specifically developed for this study that measured the use of healthcare services and the personal costs of respondents' healthcare. Results: Health anxiety was associated with more incidents of inpatient care and greater healthcare expenditure. These associations remained significant even after controlling for state anxiety, depression, obsessionality, and the presence of chronic health problems. Conclusion: We conclude that health anxiety is related to personal as well as social costs in Japan.
PMCID: PMC4346019  PMID: 25750822
Internet-based research; mental health and disorder; health anxiety; hypochondriasis; healthcare cost
16.  Long-term outcomes of common atrioventricular valve plasty in patients with functional single ventricle 
Common atrioventricular valve (CAVV) regurgitation is widely known as a risk factor for mortality and Fontan completion in patients with functional single ventricle. Hence, we reviewed our surgical experience with CAVV plasty in Fontan candidates.
Staged Fontan strategy and extracardiac total cavopulmonary connection as Fontan modification were our principal approaches in 1995. Since then, 38 consecutive Fontan candidates (21 males, median weight at operation was 7.0 kg and median age was 17 months old) underwent CAVV plasty. Right atrial isomerism was associated with 24 patients. The initial CAVV plasty was performed before inter-stage bidirectional Glenn (BDG) in 3 patients, at BDG in 23, before Fontan in 4 and during Fontan in 8. Since 1995, the modified Alfieri technique with a tailed, expanded, polytetrafluoroethylene tube as a bridging strip was the procedure for repair and 27 patients underwent the procedure. The mean follow-up period was 7.1 years (range 0–17 years).
Actuarial survival and freedom from CAVV replacement rates at 1, 5 and 10 years were 81, 70 and 67% and 89, 85 and 75%, respectively. Seven patients ultimately underwent CAVV replacement with one death. Twenty-three of the 38 patients completed Fontan operation (61%). Association with total anomalous pulmonary venous connection (P= 0.01) and CAVV plasty before BDG (P= 0.05) were risk factors for mortality.
CAVV plasty for patients with functional single ventricle is still challenging; however, the aggressive and repeated surgical intervention may contribute to provide better life-prognosis. The ventricular volume unloading effect of BDG without additional pulmonary blood flow or Fontan operation did not contribute to maintain CAVV function. Therefore, there would not be any hesitation for CAVV replacement to control CAVVR in the setting of systemic ventricular failure. Although the statistically significant therapeutic superiority of the modified Alfieri technique was not shown so far, further follow-up may reveal the advantage of this easy and simple technique.
PMCID: PMC3930217  PMID: 24336698
Single ventricle; Fontan; Common atrioventricular valve; Atrioventricular valve plasty
17.  Treatment of Leptothrix Cells with Ultrapure Water Poses a Threat to Their Viability 
Biology  2015;4(1):50-66.
The genus Leptothrix, a type of Fe/Mn-oxidizing bacteria, is characterized by its formation of an extracellular and microtubular sheath. Although almost all sheaths harvested from natural aquatic environments are hollow, a few chained bacterial cells are occasionally seen within some sheaths of young stage. We previously reported that sheaths of Leptothrix sp. strain OUMS1 cultured in artificial media became hollow with aging due to spontaneous autolysis within the sheaths. In this study, we investigated environmental conditions that lead the OUMS1 cells to die. Treatment of the cells with ultrapure water or acidic buffers (pH 6.0) caused autolysis of the cells. Under these conditions, the plasma membrane and cytoplasm of cells were drastically damaged, resulting in leakage of intracellular electrolytes and relaxation of genomic DNA. The autolysis was suppressed by the presence of Ca2+. The hydrolysis of peptidoglycan by the lysozyme treatment similarly caused autolysis of the cells and was suppressed also by the presence of Ca2+. However, it remains unclear whether the acidic pH-dependent autolysis is attributable to damage of peptidoglycan. It was observed that L. discophora strain SP-6 cells also underwent autolysis when suspended in ultrapure water; it is however, uncertain whether this phenomenon is common among other members of the genus Leptothrix.
PMCID: PMC4381217  PMID: 25634812
Leptothrix; cell death induced by UPW; acidic pH to cell viability; lack of Ca2+ to cell viability; peptidoglycan; damage of bacterial membrane; relaxation of DNA; electrolytes leakage
18.  Biomechanical Evaluation of Ti-Nb-Sn Alloy Implants with a Low Young’s Modulus 
Dental implants are widely used and are a predictable treatment in various edentulous cases. Occlusal overload may be causally related to implant bone loss and a loss of integration. Stress concentrations may be diminished using a mechanobiologically integrated implant with bone tissue. The purpose of this study was to investigate the biomechanical behavior, biocompatibility and bioactivity of a Ti-Nb-Sn alloy as a dental implant material. It was compared with cpTi. Cell proliferation and alkaline phosphatase (ALP) activity were quantified. To assess the degree of osseointegration, a push-in test was carried out. Cell proliferation and ALP activity in the cells grown on prepared surfaces were similar for the Ti-Nb-Sn alloy and for cpTi in all the experiments. A comparison between the Ti-Nb-Sn alloy implant and the cpTi implant revealed that no significant difference was apparent for the push-in test values. These results suggest that implants fabricated using Ti-Nb-Sn have a similar biological potential as cpTi and are capable of excellent osseointegration.
PMCID: PMC4394505  PMID: 25775158
low Young’s modulus; Ti-Nb-Sn alloy; implant
19.  Cancer/Testis Antigen PAGE4, a Regulator of c-Jun Transactivation, Is Phosphorylated by Homeodomain-Interacting Protein Kinase 1, a Component of the Stress-Response Pathway 
Biochemistry  2014;53(10):1670-1679.
Prostate-associated gene 4 (PAGE4) is a cancer/testis antigen that is typically restricted to the testicular germ cells but is aberrantly expressed in cancer. Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the developing prostate and is also a stress-response protein that is upregulated in response to cellular stress. PAGE4 interacts with c-Jun, which is activated by the stress-response kinase JNK1, and plays an important role in the development and pathology of the prostate gland. Here, we have identified homeodomain-interacting protein kinase 1 (HIPK1), also a component of the stress-response pathway, as a kinase that phosphorylates PAGE4 at T51. We show that phosphorylation of PAGE4 is critical for its transcriptional activity since mutating this T residue abolishes its ability to potentiate c-Jun transactivation. In vitro single molecule FRET indicates phosphorylation results in compaction of (still) intrinsically disordered PAGE4. Interestingly, however, while our previous observations indicated that the wild-type nonphosphorylated PAGE4 protein interacted with c-Jun [RajagopalanK. et al. (2014) Biochim, Biophys. Acta1842, 154−16324263171], here we show that phosphorylation of PAGE4 weakens its interaction with c-Jun in vitro. These data suggest that phosphorylation induces conformational changes in natively disordered PAGE4 resulting in its decreased affinity for c-Jun to promote interaction of c-Jun with another, unidentified, partner. Alternatively, phosphorylated PAGE4 may induce transcription of a novel partner, which then potentiates c-Jun transactivation. Regardless, the present results clearly implicate PAGE4 as a component of the stress-response pathway and uncover a novel link between components of this pathway and prostatic development and disease.
PMCID: PMC4198062  PMID: 24559171
20.  Correlation of Sprouty1 and Jagged1 With Aggressive Prostate Cancer Cells With Different Sensitivities to Androgen Deprivation 
Journal of cellular biochemistry  2014;115(9):1505-1515.
Prostate cancer is a heterogeneous disease and thus, it is important to understand whether among the heterogeneous collection of cell types, androgen-deprivation insensitive cells exist prior to hormonal manipulation. We established several LNCaP subclones with distinct insensitivities to androgen deprivation from a parental LNCaP cell line. In the resulting clones, the sensitivity to androgen-deprivation negatively correlated with their PSA expression levels. In two of these clones, an androgen insensitive clone, LNCaP-cl1, and an androgen sensitive clone, LNCaP-cl5, the DNA copy number differed significantly, indicating that these clones contain genetically distinct cells. LNCaP-cl1 had higher PSA expression but lower invasiveness and tumor growth potential than LNCaP-cl5. The expression levels of two genes that are known to be regulated by miR-21, an androgen-regulated microRNA, Sprouty1 (SPRY1) and Jagged1 (JAG1) were significantly lower in LNCaP-cl1 than in LNCaP-cl5. Knocking down SPRY1 in LNCaP cells enhanced PSA expression and cell proliferation. JAG1 administration in LNCaP cells enhanced cell invasion and JAG1 knockdown in PC3 cells suppressed cell invasion and tumor formation. These results indicated that the expression differences in SPRY1 and JAG1 may contribute to the phenotypic differences between the LNCaP-cl1 and LNCaP-cl5 clones. In tissue samples, SPRY1 expression levels were significantly lower in prostate cancer patients with PSA recurrence after surgical treatment (P = 0.0076) and JAG1 expression levels were significantly higher in Gleason sum (GS) 8–9 disease than in GS 5–6 (P = 0.0121). In summary a random population of LNCaP cells comprises a heterogeneous group of cells with different androgen-deprivation sensitivities and potential for invasiveness.
PMCID: PMC4337863  PMID: 24604720
21.  A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus 
Nucleic Acids Research  2015;43(5):2853-2863.
DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3′ from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5′ from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea.
PMCID: PMC4357722  PMID: 25694513
22.  Development of a method for reconstruction of crowded NMR spectra from undersampled time-domain data 
Journal of Biomolecular Nmr  2015;62(1):31-41.
NMR is a unique methodology for obtaining information about the conformational dynamics of proteins in heterogeneous biomolecular systems. In various NMR methods, such as transferred cross-saturation, relaxation dispersion, and paramagnetic relaxation enhancement experiments, fast determination of the signal intensity ratios in the NMR spectra with high accuracy is required for analyses of targets with low yields and stabilities. However, conventional methods for the reconstruction of spectra from undersampled time-domain data, such as linear prediction, spectroscopy with integration of frequency and time domain, and analysis of Fourier, and compressed sensing were not effective for the accurate determination of the signal intensity ratios of the crowded two-dimensional spectra of proteins. Here, we developed an NMR spectra reconstruction method, “conservation of experimental data in analysis of Fourier” (Co-ANAFOR), to reconstruct the crowded spectra from the undersampled time-domain data. The number of sampling points required for the transferred cross-saturation experiments between membrane proteins, photosystem I and cytochrome b6f, and their ligand, plastocyanin, with Co-ANAFOR was half of that needed for linear prediction, and the peak height reduction ratios of the spectra reconstructed from truncated time-domain data by Co-ANAFOR were more accurate than those reconstructed from non-uniformly sampled data by compressed sensing.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-015-9908-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4432090  PMID: 25677224
Sparse sampling; Non-uniform sampling; Transferred cross-saturation; Membrane proteins
23.  Establishment of new predictive markers for distant recurrence of colorectal cancer using lectin microarray analysis 
Cancer Medicine  2014;4(2):293-302.
We evaluated the clinical benefits of novel predictive markers for distant recurrence with colorectal cancer using lectin microarray analysis of cell surface glycan modifications. Glycoproteins were extracted from formalin-fixed, paraffin-embedded tumor specimens and normal epithelium from 53 consecutive curatively resected stage I–III colorectal cancer cases and then subjected to lectin microarray to obtain lectin–glycan interaction (LGI) values. In addition, clinicopathological factors associated with distant recurrence were identified. LGI values that were associated with distant recurrence were validated with an additional 55 curatively resected stage II colorectal cancer cases. LGI values for Agaricus bisporus (ABA) lectin, prominent in cancer tissues, were statistically associated with distant recurrence. ABA lectin staining exhibited strikingly intense signals in the cytoplasm and apical surfaces of cancer cells, while weak staining was observed in the supranuclear regions of normal epithelium. This ABA tumor/normal LGI ratio may be a new predictive biomarker for distant recurrence of curatively resected colorectal cancer.
PMCID: PMC4329012  PMID: 25355679
Colorectal cancer; glycoprotein; lectin; prediction; recurrence
24.  99mTc-GSA SPECT/CT fused images for assessment of hepatic function and hepatectomy planning 
To assure safe hepatectomy, accurate estimation of the functional reserve of the future remnant liver is crucial. The combination of indocyanine green retention rate at 15 min and CT volumetry is widely used in deciding on the extent of hepatectomy. On the other hand, there are quantitative indices calculated from 99mTc-GSA scintigraphy that reflect the number and function of hepatocytes. Therefore, there are many indices calculated from 99mTc-GSA scintigraphy that have been reported. In recent Annals of Surgical Oncology on Oct. 2014 the Uptake Index (UI) calculated from 99mTc-GSA scintigraphy was reported to be useful for hepatectomy planning and postoperative liver failure prediction. In this paper, we report on the usefulness and limits of quantitative indices calculated from 99mTc-GSA SPECT/CT for preoperative simulation.
PMCID: PMC4322173  PMID: 25738137
99mTc-GSA SPECT/CT fused image; Uptake Index (UI); hepatectomy planning
25.  Embolization of Spontaneous Intratumoral Hemorrhage with the Hemodynamic Characteristics of Arteriovenous Fistula in Renal Angiomyolipoma 
Aneurysms within renal angiomyolipomas (AML) may rupture into the tumor or pararenal space. Transcatheter arterial embolization is the first-choice treatment to control bleeding. Here, we describe the use of coil embolization in two cases of spontaneous intratumoral hemorrhage with the hemodynamic characteristics of renal arteriovenous (AV) fistula in renal AML. In case 1, renal angiography showed several intratumoral aneurysms, one of which had ruptured into the tumor, resulting in the formation of an intratumoral hematoma. Blood flow within the hematoma was rapid and the blood was immediately returned to the systemic circulation through the left renal vein. In case 2, renal angiography showed that the rupture of an intratumoral aneurysm of a tumor-feeding artery had resulted in formation of an intratumoral hematoma and direct renal vein communication. No extratumoral hemorrhage was observed in either case. The hemodynamics of both hematomas resembled those of a high-flow renal AV fistula. The ruptured aneurysms were embolized with detachable and pushable coils (case 1) or pushable coils only (case 2). To our knowledge, this is the first report of successful embolization of AV fistula-like intratumoral hemorrhage in renal AML.
PMCID: PMC4360528  PMID: 25789276
Aneurysm; Angiomyolipoma; Embolization; Kidney; Rupture

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