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1.  Long-Term Pancreatic Beta Cell Exposure to High Levels of Glucose but Not Palmitate Induces DNA Methylation within the Insulin Gene Promoter and Represses Transcriptional Activity 
PLoS ONE  2015;10(2):e0115350.
Recent studies have implicated epigenetics in the pathophysiology of diabetes. Furthermore, DNA methylation, which irreversibly deactivates gene transcription, of the insulin promoter, particularly the cAMP response element, is increased in diabetes patients. However, the underlying mechanism remains unclear. We aimed to investigate insulin promoter DNA methylation in an over-nutrition state. INS-1 cells, the rat pancreatic beta cell line, were cultured under normal-culture-glucose (11.2 mmol/l) or experimental-high-glucose (22.4 mmol/l) conditions for 14 days, with or without 0.4 mmol/l palmitate. DNA methylation of the rat insulin 1 gene (Ins1) promoter was investigated using bisulfite sequencing and pyrosequencing analysis. Experimental-high-glucose conditions significantly suppressed insulin mRNA and increased DNA methylation at all five CpG sites within the Ins1 promoter, including the cAMP response element, in a time-dependent and glucose concentration-dependent manner. DNA methylation under experimental-high-glucose conditions was unique to the Ins1 promoter; however, palmitate did not affect DNA methylation. Artificial methylation of Ins1 promoter significantly suppressed promoter-driven luciferase activity, and a DNA methylation inhibitor significantly improved insulin mRNA suppression by experimental-high-glucose conditions. Experimental-high-glucose conditions significantly increased DNA methyltransferase activity and decreased ten-eleven-translocation methylcytosine dioxygenase activity. Oxidative stress and endoplasmic reticulum stress did not affect DNA methylation of the Ins1 promoter. High glucose but not palmitate increased ectopic triacylglycerol accumulation parallel to DNA methylation. Metformin upregulated insulin gene expression and suppressed DNA methylation and ectopic triacylglycerol accumulation. Finally, DNA methylation of the Ins1 promoter increased in isolated islets from Zucker diabetic fatty rats. This study helps to clarify the effect of an over-nutrition state on DNA methylation of the Ins1 promoter in pancreatic beta cells. It provides new insights into the irreversible pathophysiology of diabetes.
PMCID: PMC4319953  PMID: 25658116
2.  Progressive Relapse of Ligamentum Flavum Ossification Following Decompressive Surgery 
Asian Spine Journal  2014;8(6):835-839.
Thoracic ossification of the ligamentum flavum (T-OLF) is a relatively rare spinal disorder that generally requires surgical intervention, due to its progressive nature and the poor response to conservative therapy. The prevalence of OLF has been reported at 3.8%-26%, which is similar to that of cervical ossification of the posterior longitudinal ligament (OPLL). The progression of OPLL after cervical laminoplasty for the treatment of OPLL is often shown in long-term follow-up. However, there have been no reports on the progression of OLF following surgery. We report a case of thoracic myelopathy secondary to the progressive relapse of OLF following laminectomy.
PMCID: PMC4278992  PMID: 25558329
Thoracic ossification of ligamentum flavum; Progressive relapse
3.  Comparing Biofouling Control Treatments for Use on Aquaculture Nets 
Test panels comprised of uncoated, copper coated and silicone coated 7/8'' (22 mm) mesh knitted nylon net were evaluated to compare their properties and the effectiveness to prevent biofouling. This paper describes test procedures that were developed to quantify the performance in terms of antifouling, cleanability, drag and cost. The copper treatment was the most effective at controlling fouling, however, the silicone treated nets were the easiest to clean. The drag forces on the net were a function of twine diameter, twine roughness and fouling. After immersion, the uncoated nets had the most drag followed by the silicone and copper treatments. The cost of applying silicone to nets is high; however, improved formulations may provide a non-toxic alternative to control fouling.
PMCID: PMC4284699  PMID: 25474085
net fouling; net coatings; drag; biocides; fouling release; cleaning; test methods
4.  Hypoxia and fatty liver 
World Journal of Gastroenterology : WJG  2014;20(41):15087-15097.
The liver is a central organ that metabolizes excessive nutrients for storage in the form of glycogen and lipids and supplies energy-producing substrates to the peripheral tissues to maintain their function, even under starved conditions. These processes require a considerable amount of oxygen, which causes a steep oxygen gradient throughout the hepatic lobules. Alcohol consumption and/or excessive food intake can alter the hepatic metabolic balance drastically, which can precipitate fatty liver disease, a major cause of chronic liver diseases worldwide, ranging from simple steatosis, through steatohepatitis and hepatic fibrosis, to liver cirrhosis. Altered hepatic metabolism and tissue remodeling in fatty liver disease further disrupt hepatic oxygen homeostasis, resulting in severe liver hypoxia. As master regulators of adaptive responses to hypoxic stress, hypoxia-inducible factors (HIFs) modulate various cellular and organ functions, including erythropoiesis, angiogenesis, metabolic demand, and cell survival, by activating their target genes during fetal development and also in many disease conditions such as cancer, heart failure, and diabetes. In the past decade, it has become clear that HIFs serve as key factors in the regulation of lipid metabolism and fatty liver formation. This review discusses the molecular mechanisms by which hypoxia and HIFs regulate lipid metabolism in the development and progression of fatty liver disease.
PMCID: PMC4223242  PMID: 25386057
Hypoxia; Fatty liver disease; Hypoxia-inducible factor; Lipid metabolism; Obstructive sleep apnea
5.  Long unidirectional barbed suturing technique with extracorporeal traction in laparoscopic myomectomy 
BMC Surgery  2014;14(1):84.
Myomectomy is now often performed laparoscopically rather than by laparotomy to alleviate the risk of postoperative adhesions and reduce postoperative pain. However, intracorporeal knot-tying under direct laparoscopy is difficult and requires proficiency. We conducted a retrospective study comparing the results of a long unidirectional barbed suturing technique (with V-Loc180 suture) and the results of conventional suturing as applied to laparoscopic myomectomy.
In women who underwent laparoscopic myomectomy in our university hospital between January 2011 and April 2013, uninterrupted suturing of 2 or more layers was performed. These women were divided into 2 groups according to the method of suturing: those in whom standard absorbable sutures were used (group P, n =42) and those in whom our suturing technique was used (group V, n =41). Patient characteristics and surgical variables were compared between the 2 groups.
No significant between-group difference was observed in age (p = .975), body mass index (p = .778), GnRHa administration (p = .059), intraoperative vasopressin dose (p = .364), intraoperative blood loss (73.8 ± 64.1 vs. 59.3 ± 54.0 mL, respectively; p = .199), myoma mass (212.6 ± 133.3 vs. 208.3 ± 198.4 g, respectively; p = .134), ΔHb (p = .517), or postoperative hospital stay (p = .314). Operation time (mean ± SD) was significantly shorter for group V (71.2 ± 22.9 minutes; range, 28.0–110.0 minutes; p < .001) than for group P (94.4 ± 27.2 minutes; range, 53.0–165.0 minutes). No patient required intraoperative transfusion or conversion to laparotomy.
Our suturing technique exploits the features of unidirectional barbed sutures and can be used in the same way as the conventional method when performing continuous suturing for laparoscopic myomectomy. Our data suggest that operation time can be reduced by as much as 25% with this new technique.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2482-14-84) contains supplementary material, which is available to authorized users.
PMCID: PMC4217315  PMID: 25345546
6.  Safety and possible effects of low-intensity resistance training associated with partial blood flow restriction in polymyositis and dermatomyositis 
Our aim was to evaluate the safety and efficacy of a low-intensity resistance training program combined with partial blow flow restriction (BFR training) in a cohort of patients with polymyositis (PM) and dermatomyositis (DM).
In total, 13 patients with PM and DM completed a 12-week twice a week low-intensity (that is, 30% one-repetition-maximum (1RM)) resistance exercise training program combined with partial blood flow restriction (BFR). Assessments of muscle strength, physical function, quadriceps cross sectional (CSA) area, health-related quality of life, and clinical and laboratory parameters were assessed at baseline and after the intervention.
The BFR training program was effective in increasing the maximal dynamic strength in both the leg-press (19.6%, P <0.001) and knee-extension exercises (25.2% P <0.001), as well as in the timed-stands (15.1%, P <0.001) and timed-up-and-go test (−4.5%, P =0.002). Quadriceps CSA was also significantly increased after the intervention (4.57%, P =0.01). Similarly, all of the components of the Short Form-36 Health Survey, the Health Assessment Questionnaire scores, and the patient- and physician reported Visual Analogue Scale were significantly improved after training (P <0.05). Importantly, no clinical evidence or any other self-reported adverse event were found. Laboratory parameters (creatine kinase and aldolase) were also unchanged (P >0.05) after the intervention.
We demonstrated that a 12-week supervised low-intensity resistance training program associated with partial blood flow restriction may be safe and effective in improving muscle strength and function as well as muscle mass and health-related quality of life in patients with PM and DM.
Trial registration NCT01501019. Registered November 29, 2011.
PMCID: PMC4232679  PMID: 25344395
7.  Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies 
Science translational medicine  2014;6(224):224ra24.
The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.
PMCID: PMC4017867  PMID: 24553385
8.  Transient Helical Structure during PI3K and Fyn SH3 Domain Folding 
The journal of physical chemistry. B  2013;117(17):10.1021/jp400167s.
A growing list of proteins, including the β-sheet-rich SH3 domain, is known to transiently populate a compact α-helical intermediate before settling into the native structure. Examples have been discovered in cryogenic solvent as well as by pressure jumps. Earlier studies of lambda repressor mutants showed that transient states with excess helix are robust in an all-α protein. Here we extend a previous study of src SH3 domain to two new SH3 sequences, phosphatidylinositol 3-kinase (PI3K) and a Fyn mutant, to see how robust such helix-rich transients are to sequence variations in this β-sheet fold. We quantify helical structure by circular dichroism (CD), protein compactness by small angle X-ray scattering (SAXS), and transient helical populations by cryo-stopped flow CD. Our results show that transient compact helix-rich intermediates are easily accessible on the folding landscape of different SH3 domains. In molecular dynamics simulations, force field errors are often blamed for transient non-native structure. We suggest that experimental examples of very fast α-rich transient misfolding could become a more subtle test for further force field improvements than observation of the native state alone.
PMCID: PMC3841067  PMID: 23537292
circular dichroism; small angle X-ray scattering; cryosolvent; secondary structure; protein folding
9.  Identification of causative pregnancy of gestational trophoblastic neoplasia diagnosed during pregnancy by short tandem repeat analysis 
•Gestational trophoblastic neoplasia can arise during the first trimester originating from trophoblasts of concurrent pregnancy.•Intraplacental choriocarcinoma can be developed from trophoblasts of a previous pregnancy.
PMCID: PMC4059915  PMID: 24944881
DNA analysis; Intra-placental choriocarcinoma; Placental site trophoblastic tumor; Pregnancy; Short tandem repeat
10.  Incidence and Clinical Impact of Stent Fracture After the Nobori Biolimus‐Eluting Stent Implantation 
Stent fracture (SF) after drug‐eluting stent implantation has become an important concern. The aim of this study was to assess the incidence, predictors, and clinical impact of SF after biolimus‐eluting stent.
Methods and Results
A total of 1026 patients with 1407 lesions undergoing the Nobori biolimus‐eluting stent implantation and follow‐up angiography within 9 months after index procedure were analyzed. SF was defined as complete or partial separation of the stent, as assessed by using plain fluoroscopy, intravascular ultrasound, or optical coherence tomography during the follow‐up. We assessed the rate of SF and the cumulative incidence of clinically driven target lesion revascularization and definite stent thrombosis within 9 months. SF was observed in 58 (4.1%) of 1407 lesions and 57 (5.5%) of 1026 patients. Lesions with hinge motion (OR 8.90, 95% CI 3.84 to 20.6, P<0.001), tortuosity (OR 4.16, 95% CI 1.75 to 9.88, P=0.001), and overlapping stents (OR 2.41, 95% CI 0.95 to 6.10, P=0.06) were predictors of SF. Cumulative incidence of clinically driven target lesion revascularization within 9 months was numerically higher in the SF group than that in the non‐SF group (12.0% versus 1.0%). Cumulative incidence of definite stent thrombosis within 9 months tended to be higher in the SF group than that in the non‐SF group (1.7% versus 0.5%).
SF after biolimus‐eluting stent occurs in 4.1% of lesions and appears to be associated with clinically driven target lesion revascularization.
PMCID: PMC4187487  PMID: 24650925
biolimus‐eluting stent; drug‐eluting stent fracture; in‐stent restenosis; percutaneous coronary intervention
11.  Structural study of hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering 
Biophysical chemistry  2013;0:39-46.
We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer–dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer–dimer transition. This monomer–dimer transition was accompanied with helix-β transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine β-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.
PMCID: PMC3925460  PMID: 23524290
Equilibrium helix-rich intermediate; Monomer–dimer transition; C-state
12.  Unilateral instrumented fixation for cervical dumbbell tumors 
The purpose of this study was to describe the radiological outcomes in patients with unilateral instrumented fixation for cervical dumbbell tumors.
Patients and methods
Fourteen consecutive individuals were included in the present study. We included Eden type II and III tumors in this cohort study and analyzed fixed segment fusion rates, screw failure with multiplanar reconstruction computed tomography (CT) scan radiographs and lateral radiographs with flexion-extension dynamic views, and immediate postoperative and last follow-up radiographs after surgery.
The mean follow-up was 105.4 months. There were six men and eight women ranging in age from 32 to 70 years (mean age, 48 years). Twenty pedicle screws (PSs) and 11 lateral mass screws (LMSs) were used in total. There were seven patients with only PSs, four with only LMSs, and three with PSs at C2 and LMSs at C3. PS misplacement occurred in three screws of insertions including two screws with grade 1 misplacement and one screw with grade 2 misplacement, and no grade 3 misplacement occurred. All screws breached the lateral wall with no apparent superior or inferior misplacement. None of the LMSs were misplaced. Fortunately, no complication could be directly attributed to screw insertion. Radiological evidence showed that all patients achieved successful fusion with no screw loosening or breakage. However, two patients who received only LMS fixation had degenerative spondylolisthesis at the upper fusion segment at the last follow-up.
Grade 2 PS misplacement occurred in one screw of insertions. Unilateral pedicle screw fixation for cervical dumbbell tumors is a useful surgical method that can successfully fuse vertebrae with good postoperative alignment.
PMCID: PMC3898569  PMID: 24438086
Unilateral instrumented fixation; Cervical dumbbell tumors; Pedicle screw misplacement
13.  High expression of N-acetylglucosaminyltransferase IVa promotes invasion of choriocarcinoma 
British Journal of Cancer  2012;107(12):1969-1977.
Gestational trophoblastic diseases (GTDs) are related to trophoblasts, and human chorionic gonadotropin (hCG) is secreted by GTDs as well as normal placentas. However, the asparagine-linked sugar chains on hCG contain abnormal biantennary structures in invasive mole and choriocarcinoma, but not normal pregnancy or hydatidiform mole. N-acetylglucosaminyltransferase-IV (GnT-IV) catalyses β1,4-N-acetylglucosamine branching on asparagine-linked oligosaccharides, which are consistent with the abnormal sugar chain structures on hCG.
We investigated GnT-IVa expression in GTDs and placentas by immunohistochemistry, western blot, and RT–PCR. We assessed the effects of GnT-IVa knockdown in choriocarcinoma cells in vitro and in vivo.
The GnT-IVa was highly expressed in trophoblasts of invasive mole and choriocarcinoma, and moderately in extravillous trophoblasts during the first trimester, but not in hydatidiform mole or other normal trophoblasts. The GnT-IVa knockdown in choriocarcinoma cells significantly reduced migration and invasive capacities, and suppressed cellular adhesion to extracellular matrix proteins. The extent of β1,4-N-acetylglucosamine branching on β1 integrin was greatly reduced by GnT-IVa knockdown, although the expression of β1 integrin was not changed. In vivo studies further demonstrated that GnT-IVa knockdown suppressed tumour engraftment and growth.
These findings suggest that GnT-IVa is involved in regulating invasion of choriocarcinoma through modifications of the oligosaccharide chains of β1 integrin.
PMCID: PMC3516685  PMID: 23169300
beta1 integrin; choriocarcinoma; human chorionic gonadotropin; invasion; N-acetylglucosaminyltransferase-IV
14.  Correction: Analysis of the Proteinaceous Components of the Organic Matrix of Calcitic Sclerites from the Soft Coral Sinularia sp. 
PLoS ONE  2013;8(11):10.1371/annotation/40505aa8-17cf-49c0-8d1d-1e28af8553cb.
PMCID: PMC3850287
15.  Features of the Onset of Takayasu's Arteritis According to Gender 
Arquivos Brasileiros de Cardiologia  2013;101(4):359-365.
Although there are various published epidemiological studies regarding Takayasu's arteritis (TA), none have analyzed the influence of gender on the clinical and laboratory manifestations or vascular alterations at disease onset.
To analyze the influence of gender on clinical and laboratory manifestations and variations in vascular imaging at TA onset.
A retrospective, unicentric cohort study that evaluated 55 consecutive TA patients between 1982 and 2012. All available clinical data and laboratory test results related to the onset of the disease were analyzed. We included only patients aged 12-35 years at diagnosis to exclude age-related factors.
We analyzed 17 men and 38 women, mostly Caucasian, with a comparable mean age between genders. There was no gender difference regarding the clinical or laboratory characteristics, comorbidities, or smoking habit, except for abdominal pain, which was more common in men. Regarding vascular lesions, the presence of ascending aortic aneurysms was significantly more frequent in males. Male gender represented an independent risk factor for the occurrence of abdominal pain and ascending aortic aneurysms in TA patients.
Abdominal pain and ascending aortic aneurysms occurred more frequently in men with TA, suggesting a more severe disease profile in males.
PMCID: PMC4062373  PMID: 23979780
Takayasu Arteritis / epidemiology; Male; Female; Cohort Studies; Aortic Aneurysm, Abdominal; Abdominal Pain
16.  Myositis-specific and myositis-associated autoantibody profiles and their clinical associations in a large series of patients with polymyositis and dermatomyositis 
Clinics  2013;68(7):909-914.
To analyze the prevalence of myositis-specific and myositis-associated autoantibodies and their clinical correlations in a large series of patients with dermatomyositis/polymyositis.
This cross-sectional study enrolled 127 dermatomyositis cases and 95 polymyositis cases. The disease-related autoantibody profiles were determined using a commercially available blood testing kit.
The prevalence of myositis-specific autoantibodies in all 222 patients was 34.4%, whereas myositis-associated autoantibodies were found in 41.4% of the patients. The most frequently found autoantibody was anti-Ro-52 (36.9%), followed by anti-Jo-1 (18.9%), anti-Mi-2 (8.1%), anti-Ku (4.1%), anti-SRP (3.2%), anti-PL-7 (3.2%), anti-PL-12 (2.7%), anti-PM/Scl75 (2.7%), and anti-PM/Scl100 (2.7%). The distributions of these autoantibodies were comparable between polymyositis and dermatomyositis, except for a higher prevalence of anti-Jo-1 in polymyositis. Anti-Mi-2 was more prevalent in dermatomyositis. Notably, in the multivariate analysis, anti-Mi-2 and anti-Ro-52 were associated with photosensitivity and pulmonary disorders, respectively, in dermatomyositis. Anti-Jo-1 was significantly correlated with pulmonary disorders in polymyositis. Moreover, anti-Ro-52 was associated with anti-Jo-1 in both diseases. No significant correlation was observed between the remaining autoantibodies and the clinical and/or laboratory findings.
Our data are consistent with those from other published studies involving other populations, although certain findings warrant consideration. Anti-Ro-52 and anti-Jo-1 were strongly associated with one another. Anti-Ro-52 was correlated with pulmonary disorders in dermatomyositis, whereas anti-Jo-1 was correlated with pulmonary alterations in polymyositis.
PMCID: PMC3715024  PMID: 23917652
Dermatomyositis; Idiopathic Inflammatory Myopathies; Myositis-Associated Autoantibodies; Myositis-Specific Autoantibodies; Polymyositis
17.  Chloroquine diphosphate: a risk factor for herpes zoster in patients with dermatomyositis/polymyositis 
Clinics  2013;68(5):621-627.
Herpes zoster has been widely described in the context of different systemic autoimmune diseases but not dermatomyositis/polymyositis. Therefore, we analyzed the prevalence, risk factors and herpes zoster outcomes in this population.
A retrospective cohort study of herpes zoster infections in dermatomyositis/polymyositis patients was performed. The patients were followed at a tertiary center from 1991 to 2012. For the control group, each patient with herpes zoster was paired with two patients without herpes zoster. Patients were matched by gender and the type of myositis, age at myositis onset and disease duration.
Of 230 patients, 24 (10.4%) had a histories of herpes zoster (19 with dermatomyositis and five with polymyositis, two-thirds female). The mean age of the patients with herpes zoster was 44.6±16.8 years. No difference between the groups was found regarding cumulative clinical manifestations. Disease activity, autoantibody, muscle and leukogram parameters were also comparable between the groups. No differences in immunosuppressive (alone or in association with other immunosuppressive therapies) or glucocorticoid (current use, medium dose and cumulative dose in the last two months) therapies were found between patients with and without herpes zoster. However, a higher proportion of patients in the herpes zoster group received chloroquine diphosphate compared to the control group. All of the patients received acyclovir; 58.3% of patients had postherpetic neuralgia and no cases of recurrence were reported. Furthermore, individuals who were taking high prednisone doses at the time of the herpes zoster diagnosis had reduced levels of postherpetic neuralgia.
These data suggest that chloroquine diphosphate could predispose patients with dermatomyositis/polymyositis to developing herpes zoster, particularly women and dermatomyositis patients.
PMCID: PMC3654292  PMID: 23778404
Antimalarial; Chloroquine Diphosphate; Dermatomyositis; Herpes Zoster; Inflammatory Myopathies; Polymyositis; Risk Factors
18.  Fatal pulmonary embolism in hospitalized patients: a large autopsy-based matched case-control study 
Clinics  2013;68(5):679-685.
Pulmonary embolism is an underdiagnosed major cause of death for hospitalized patients. The objective of this study was to identify the conditions associated with fatal pulmonary embolism in this population.
A total of 13,074 autopsy records were evaluated in a case-control study. Patients were matched by age, sex, and year of death, and factors potentially associated with fatal pulmonary embolism were analyzed using univariate and multivariate conditional logistic regression.
Pulmonary embolism was considered fatal in 328 (2.5%) patients. In the multivariate analysis, conditions that were more common in patients who died of pulmonary embolism were atherosclerosis, congestive heart failure, and neurological surgery. Some conditions were negatively associated with fatal pulmonary embolism, including hemorrhagic stroke, aortic aneurism, cirrhosis, acquired immune deficiency syndrome, and pneumonia. In the control group, patients with hemorrhagic stroke and aortic aneurism had short hospital stays (8.5 and 8.8 days, respectively), and the hemorrhage itself was the main cause of death in most of them (90.6% and 68.4%, respectively), which may have prevented the development of pulmonary embolism. Cirrhotic patients in the control group also had short hospital stays (7 days), and 50% died from bleeding complications.
In this large autopsy study, atherosclerosis, congestive heart failure, and neurological surgery were diagnoses associated with fatal pulmonary embolism.
PMCID: PMC3654296  PMID: 23778403
Atherosclerosis; Heart Failure; Neurosurgery; Pulmonary Embolism; Venous Thromboembolism
19.  Differential Expression of ID4 and Its Association with TP53 Mutation, SOX2, SOX4 and OCT-4 Expression Levels 
PLoS ONE  2013;8(4):e61605.
Inhibitor of DNA Binding 4 (ID4) is a member of the helix-loop-helix ID family of transcription factors, mostly present in the central nervous system during embryonic development, that has been associated with TP53 mutation and activation of SOX2. Along with other transcription factors, ID4 has been implicated in the tumorigenic process of astrocytomas, contributing to cell dedifferentiation, proliferation and chemoresistance. In this study, we aimed to characterize the ID4 expression pattern in human diffusely infiltrative astrocytomas of World Health Organization (WHO) grades II to IV of malignancy (AGII-AGIV); to correlate its expression level to that of SOX2, SOX4, OCT-4 and NANOG, along with TP53 mutational status; and to correlate the results with the clinical end-point of overall survival among glioblastoma patients. Quantitative real time PCR (qRT-PCR) was performed in 130 samples of astrocytomas for relative expression, showing up-regulation of all transcription factors in tumor cases. Positive correlation was found when comparing ID4 relative expression of infiltrative astrocytomas with SOX2 (r = 0.50; p<0.005), SOX4 (r = 0.43; p<0.005) and OCT-4 (r = 0.39; p<0.05). The results from TP53 coding exon analysis allowed comparisons between wild-type and mutated status only in AGII cases, demonstrating significantly higher levels of ID4, SOX2 and SOX4 in mutated cases (p<0.05). This pattern was maintained in secondary GBM and further confirmed by immunohistochemistry, suggesting a role for ID4, SOX2 and SOX4 in early astrocytoma tumorigenesis. Combined hyperexpression of ID4, SOX4 and OCT-4 conferred a much lower (6 months) median survival than did hypoexpression (18 months). Because both ID4 alone and a complex of SOX4 and OCT-4 activate SOX2 transcription, it is possible that multiple activation of SOX2 impair the prognosis of GBM patients. These observational results of associated expression of ID4 with SOX4 and OCT-4 may be used as a predictive factor of prognosis upon further confirmation in a larger GBM series.
PMCID: PMC3628974  PMID: 23613880
20.  Exomic Sequencing of Four Rare Central Nervous System Tumor Types 
Oncotarget  2013;4(4):572-583.
A heterogeneous population of uncommon neoplasms of the central nervous system (CNS) cause significant morbidity and mortality. To explore their genetic origins, we sequenced the exomes of 12 pleomorphic xanthoastrocytomas (PXA), 17 non-brainstem pediatric glioblastomas (PGBM), 8 intracranial ependymomas (IEP) and 8 spinal cord ependymomas (SCEP). Analysis of the mutational spectra revealed that the predominant single base pair substitution was a C:G>T:A transition in each of the four tumor types. Our data confirm the critical roles of several known driver genes within CNS neoplasms, including TP53 and ATRX in PGBM, and NF2 in SCEPs. Additionally, we show that activating BRAF mutations play a central role in both low and high grade glial tumors. Furthermore, alterations in genes coding for members of the mammalian target of rapamycin (mTOR) pathway were observed in 33% of PXA. Our study supports the hypothesis that pathologically similar tumors arising in different age groups and from different compartments may represent distinct disease processes with varied genetic composition.
PMCID: PMC3720605  PMID: 23592488
Central nervous system (CNS) tumors; cancer genetics; exome sequencing; pediatric tumors; brain tumors
21.  Analysis of the Proteinaceous Components of the Organic Matrix of Calcitic Sclerites from the Soft Coral Sinularia sp. 
PLoS ONE  2013;8(3):e58781.
An organic matrix consisting of a protein-polysaccharide complex is generally accepted as an important medium for the calcification process. While the role this “calcified organic matrix” plays in the calcification process has long been appreciated, the complex mixture of proteins that is induced and assembled during the mineral phase of calcification remains uncharacterized in many organisms. Thus, we investigated organic matrices from the calcitic sclerites of a soft coral, Sinularia sp., and used a proteomic approach to identify the functional matrix proteins that might be involved in the biocalcification process. We purified eight organic matrix proteins and performed in-gel digestion using trypsin. The tryptic peptides were separated by nano-liquid chromatography (nano-LC) and analyzed by tandem mass spectrometry (MS/MS) using a matrix-assisted laser desorption/ionization (MALDI) – time-of-flight-time-of-flight (TOF-TOF) mass spectrometer. Periodic acid Schiff staining of an SDS-PAGE gel indicated that four proteins were glycosylated. We identified several proteins, including a form of actin, from which we identified a total of 183 potential peptides. Our findings suggest that many of those peptides may contribute to biocalcification in soft corals.
PMCID: PMC3597568  PMID: 23527022
22.  Met Is the Most Frequently Amplified Gene in Endometriosis-Associated Ovarian Clear Cell Adenocarcinoma and Correlates with Worsened Prognosis 
PLoS ONE  2013;8(3):e57724.
Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p<0.05). Met knockdown by shRNA resulted in reduced viability of OCC cells with Met amplification due to increased apoptosis and cellular senescence, suggesting that the Met signaling pathway plays an important role in OCC carcinogenesis. Thus, we believe that targeted inhibition of the Met pathway may be a promising treatment for OCC.
PMCID: PMC3587638  PMID: 23469222
23.  Giant cell arteritis: a multicenter observational study in Brazil 
Clinics  2013;68(3):317-322.
To describe demographic features, disease manifestations and therapy in patients with giant cell arteritis from referral centers in Brazil.
A retrospective cohort study was performed on 45 giant cell arteritis patients from three university hospitals in Brazil. Diagnoses were based on the American College of Rheumatology classification criteria for giant cell arteritis or temporal artery biopsy findings.
Most patients were Caucasian, and females were slightly more predominant. The frequencies of disease manifestations were as follows: temporal headache in 82.2%, neuro-ophthalmologic manifestations in 68.9%, jaw claudication in 48.9%, systemic symptoms in 44.4%, polymyalgia rheumatica in 35.6% and extra-cranial vessel involvement in 17.8% of cases. Aortic aneurysms were observed in 6.6% of patients. A comparison between patients with biopsy-proven giant cell arteritis and those without temporal artery biopsies did not yield significant differences in disease manifestations. All patients were treated with oral prednisone, and intravenous methylprednisolone was administered to nearly half of the patients. Methotrexate was the most commonly used immunosuppressive agent, and low-dose aspirin was prescribed to the majority of patients. Relapses occurred in 28.9% of patients, and aspirin had a protective effect against relapses. Females had higher prevalences of polymyalgia rheumatica, systemic manifestations and jaw claudication, while permanent visual loss was more prevalent in men.
Most of the clinical features of Brazilian giant cell arteritis patients were similar to those found in other studies, except for the high prevalence of neuro-ophthalmic manifestations and permanent blindness in the Brazilian patients. Aspirin had a protective effect on relapses.
PMCID: PMC3611879  PMID: 23644850
Giant Cell Arteritis; Glucocorticoids; Methotrexate; Multicenter Study; Vasculitis
24.  Artificial multilayers and nanomagnetic materials 
The author has been actively engaged in research on nanomagnetic materials for about 50 years. Nanomagnetic materials are comprised of ferromagnetic systems for which the size and shape are controlled on a nanometer scale. Typical examples are ultrafine particles, ultrathin films, multilayered films and nano-patterned films. In this article, the following four areas of the author’s studies are described.
(1) Mössbauer spectroscopic studies of nanomagnetic materials and interface magnetism.
(2) Preparation and characterization of metallic multilayers with artificial superstructures.
(3) Giant magnetoresistance (GMR) effect in magnetic multilayers.
(4) Novel properties of nanostructured ferromagnetic thin films (dots and wires).
A subject of particular interest in the author’s research was the artificially prepared multilayers consisting of metallic elements. The motivation to initiate the multilayer investigation is described and the physical properties observed in the artificial multilayers are introduced. The author’s research was initially in the field of pure physical science and gradually extended into applied science. His achievements are highly regarded not only from the fundamental point of view but also from the technological viewpoint.
PMCID: PMC3627924  PMID: 23391605
Mössbauer spectroscopy; interface magnetism; artificial multilayers with superstructures; non-coupled type GMR (giant magnetoresistance); magnetic vortex cores; domain walls in nanomagnetic wires
25.  CTNNB1, AXIN1 and APC expression analysis of different medulloblastoma variants 
Clinics  2013;68(2):167-172.
We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatric brain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor.
Sixty-one medulloblastoma cases were analyzed for β-catenin gene (CTNNB1) mutations, β-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. All data were correlated with histological subtypes and patient clinical information.
CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3β phosphorylation sites, which participate in β-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear β-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1) were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants.
A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of β-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularity medulloblastoma variants but not in large-cell medulloblastomas.
PMCID: PMC3584274  PMID: 23525311
β-catenin; Gene Expression; Immunohistochemistry; Medulloblastoma; Wnt Pathway

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