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1.  Noncanonical Wnt5a enhances Wnt/β-catenin signaling during osteoblastogenesis 
Scientific Reports  2014;4:4493.
Wnt regulates bone formation through β-catenin-dependent canonical and -independent noncanonical signaling pathways. However, the cooperation that exists between the two signaling pathways during osteoblastogenesis remains to be elucidated. Here, we showed that the lack of Wnt5a in osteoblast-lineage cells impaired Wnt/β-catenin signaling due to the reduced expression of Lrp5 and Lrp6. Pretreatment of ST2 cells, a stromal cell line, with Wnt5a enhanced canonical Wnt ligand-induced Tcf/Lef transcription activity. Short hairpin RNA-mediated knockdown of Wnt5a, but not treatment with Dkk1, an antagonist of Wnt/β-catenin signaling, reduced the expression of Lrp5 and Lrp6 in osteoblast-lineage cells under osteogenic culture conditions. Osteoblast-lineage cells from Wnt5a-deficient mice exhibited reduced Wnt/β-catenin signaling, which impaired osteoblast differentiation and enhanced adipocyte differentiation. Adenovirus-mediated gene transfer of Lrp5 into Wnt5a-deficient osteoblast-lineage cells rescued their phenotypic features. Therefore, Wnt5a-induced noncanonical signaling cooperates with Wnt/β-catenin signaling to achieve proper bone formation.
PMCID: PMC3967152  PMID: 24670389
2.  Dynamic Sensory Representations in the Olfactory Bulb: Modulation by Wakefulness and Experience 
Neuron  2012;76(5):962-975.
How are sensory representations in the brain influenced by the state of an animal? Here we use chronic two-photon calcium imaging to explore how wakefulness and experience shape odor representations in the mouse olfactory bulb. Comparing the awake and anesthetized state, we show that wakefulness greatly enhances the activity of inhibitory granule cells and makes principal mitral cell odor responses more sparse and temporally dynamic. In awake mice, brief repeated odor experience leads to a gradual and long-lasting (months) weakening of mitral cell odor representations. This mitral cell plasticity is odor-specific, recovers gradually over months and can be repeated with different odors. Furthermore, the expression of this experience-dependent plasticity is prevented by anesthesia. Together, our results demonstrate the dynamic nature of mitral cell odor representations in awake animals, which is constantly shaped by recent odor experience.
PMCID: PMC3523713  PMID: 23217744
3.  Sox9 is upstream of microRNA-140 in cartilage 
MicroRNA-140 (miR-140) is Specifically expressed in developing cartilage tissues. We have previously reported that miR-140 plays an important role during palatal cartilage development by modulating platalet-derived growth factor receptor alpha (pdgfra) in zebrafish. However, the regulatory mechanism of miR-140 in cartilage is still unknown. Using developing zebrafish, Sox9a mutant (Sox9a−/−) and Sox9b mutant (Sox9b−/−) zebrafish and Sox9 siRNA in human chondrocytes, T/C-28 Cells, we found that miR-140 is regulated by the cartilage master transcription regulator Sox9 in zebrafish and mammalian cells.
PMCID: PMC3774128  PMID: 22052544
microRNA-140 (miR-140); Sox9; cartilage; zebrafish; T/C-28; SiRNA; in situ hybridization; RT-PCR
4.  Frequency of elevated biomarkers in patients with cryptogenic hepatocellular carcinoma 
The incidence of hepatocellular carcinoma (HCC) continues to increase in Japan, but the clinical characteristics of Japanese patients with HCC have not been well described. The aim of this study was to determine the frequencies and utilities of elevated α-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) levels as biomarkers in cryptogenic HCC.
A total of 2638 patients with HCC diagnosed between 1999 and 2010 in the Nagasaki Association Study of Liver (NASLD) were recruited for this study. The cause of HCC was categorized into 4 groups; HCC-B, HCC-C, HCC-BC, and HCC-nonBC. The significance of factors was examined for HCC-nonBC using logistic regression analysis in all patients.
Multivariate analysis identified age, sex, BMI, alcohol consumption, platelet count, AST, ALT, AFP, DCP, and TNM stage as independent and significant risk factors for HCC-nonBC. According to TNM stage, the median AFP levels in HCC-nonBC with TNM stages I, II, and III were significantly lower than in either HCC-B or HCC-C. In TNM stage IV, the median AFP level in HCC-nonBC was significantly lower than in either HCC-B or HCC-BC. The median DCP levels in HCC-nonBC with TNM stages I and II were significantly higher than those in either HCC-B or HCC-C. In TNM stage III, the median DCP level in HCC-nonBC was significantly higher than that in HCC-C.
DCP was more sensitive than AFP for the diagnosis of early stage cryptogenic HCC. DCP should be used as the main serum test for cryptogenic HCC detection.
PMCID: PMC3775616  PMID: 24008520
6.  Preoperative radiographic and histopathologic evaluation of central chondrosarcoma 
Distinguishing grade 1 chondrosarcoma from grade 2 chondrosarcoma is critical both for planning the surgical procedure and for predicting the outcome. We aimed to review the preoperative radiographic and histologic findings, and to evaluate the reliability of preoperative grading.
We retrospectively reviewed the medical records of 17 patients diagnosed with central chondrosarcoma at our institution between 1996 and 2011. In these cases, we compared the preoperative and postoperative histologic grades, and evaluated the reliability of the preoperative histologic grading. We also assessed the preoperative radiographic findings obtained using plain radiography, computed tomography (CT), and magnetic resonance imaging (MRI).
Preoperative histologic grade was 1 in 12 patients, 2 in 4 patients, and 3 in 1 patient. However, 6 of the 12 cases classified as grade 1 before surgery were re-classified as grade 2 postoperatively. In the radiographic evaluation, grade 1 was suspected by the presence of a ring-and-arc pattern of calcification on plain radiography and CT and entrapped fat and ring-and-arc enhancement on MRI. Grades 2 and 3 were suspected by the absence of calcification and the presence of cortical penetration and endosteal scalloping on plain radiography and CT, as well as soft-tissue mass formation on MRI.
Although the combination of radiographic interpretation and histologic findings may improve the accuracy of preoperative grading in chondrosarcoma, the establishment of a standard evaluation system with the histologic and radiographic findings and/or the development of new biologic markers are necessary for preoperative discrimination of low-grade chondrosarcoma from high-grade chondrosarcoma.
PMCID: PMC3751216  PMID: 23820853
Chondrosarcoma; Imaging features; Histopathology; Surgical staging
7.  Clinical validation of the gastrointestinal NET grading system: Ki67 index criteria of the WHO 2010 classification is appropriate to predict metastasis or recurrence 
Diagnostic Pathology  2013;8:65.
In the WHO 2010 classification, the neuroendocrine tumors (NETs) are subdivided by their mitotic index or Ki67 index into either G1 or G2 NETs. Tumors with a Ki67 index of <2% are classified as G1 and those with 3—20% are classified as G2. However, the assessment of tumors with Ki67 index of greater than 2% and less than or equal to 3% is still unclear. To resolve the problem, we validated the Ki67 index criteria of gastrointestinal NETs of the WHO 2010 classification.
The medical records of 45 patients who were pathologically diagnosed as having NET G1/G2 of the gastrointestinal tract were analyzed retrospectively. According to the WHO 2010 classification, Ki67 index were calculated. Computer-assisted cytometrical analysis of Ki67 immunoreactivity was performed using the WinRooF image processing software. Receiver operating characteristic (ROC) curves were generated to determine the best discriminating Ki67 index. To clarify the assessment of tumors with Ki67 index between 2—3%, the calculated cutoff of Ki67 index was evaluated using Fisher’s exact test.
ROC curve analysis confirmed that 2.8% was the best Ki67 index cutoff value for predicting metastasis or recurrence. The sensitivity of the new Ki67 index cutoff was 42.9%, and the specificity was 86.8%.
Division of NETs into G1/G2 based on Ki67 index of 3% was appropriate to predict metastases or recurrences. The WHO grading system may be the most useful classification to predict metastases or recurrences.
Virtual Slides
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PMCID: PMC3649937  PMID: 23607525
9.  Stable reconstruction using halo vest for unstable upper cervical spine and occipitocervical instability 
European Spine Journal  2011;21(2):295-303.
Upper cervical or occipitocervical disorders such as rheumatoid arthritis present as atlantoaxial subluxation, vertical subluxation of the axis, and subaxial subluxation, which produce myelopathy and severe pain. In such cases, occipitocervical reconstruction surgery may be indicated, and several reports have described reduction of subluxation by fixing the halo vest before this surgery.
The purpose of this study was to evaluate the efficacy of using the halo vest before the surgery for unstable upper cervical spine and for occipitocervical instability.
Twenty-eight patients (9 men and 19 women; mean age, 61.8 years at surgery) who presented with atlantoaxial or occipitocervical fusion were studied. In all cases, the halo vest was fixed in the conscious condition, and subluxation was reduced before the surgery. The mean follow-up period was 45 months. Roentgenologic measurement and clinical evaluation were performed before the surgery and at the final follow-up.
Using the halo vest resulted in significant reductions in the atlantodental interval, the space available for the spinal cord, and the Ranawat value (p < 0.05), and these were maintained until the final follow-up. The mean Japanese Orthopedic Association score significantly improved from 9.5 before surgery to 12.2 at the final follow-up (p = 0.01). Nineteen cases (68%) improved by more than 1 grade by Ranawat’s classification after surgery and 16 cases (57%) maintained the same at the follow-up visit.
Conscious preoperative reduction using the halo vest for occipitocervical disorders is a useful and safe technique.
PMCID: PMC3265589  PMID: 21833572
Occipitocervical disorders; Occipitocervical reconstruction; Halo vest
10.  Analysis of the anatomic subsites, gender and age in unresectable advanced colorectal carcinomas in Tochigi, Japan suggests a shift in location towards the right side colon in elderly patients treated with cetuximab 
Molecular and Clinical Oncology  2013;1(2):291-296.
Colorectal cancer is a frequently occurring cancer whose incidence has shown a marked increase in recent years. Additionally, an increase in right side colon in elderly patients has been identified. Therefore, a clinicopathological study was conducted in 49 patients with unresectable advanced colorectal carcinomas to elucidate the association of clinicopathological characteristics and K-ras mutation. Of the 49 patients included in this study, 24 were aged <60 years with a male/female (M/F) ratio of 16/8 and 25 patients were aged ≥60 years with a M/F ratio of 16/9. Of the patients aged ≥65 years, 15 patients were enrolled as controls and the M/F ratio was 9/6. Results revealed that with regard to the subsite of cancer, unresectable advanced colorectal carcinomas developed in the right-sided colon in 13 patients, left-sided colon in 19 patients and rectum in 17 patients. Right-sided colon carcinomas were commonly identified in the elderly patients aged ≥65 years, with a marked tendency in the female patients (P=0.024). Immunostaining was performed for the epidermal growth factor receptor (EGFR) antibody in 40 patients to determine whether the K-ras gene would yield positive results. The mutant K-ras gene was identified in 8 patients (20%) and the frequency was lower compared with that of the normal colorectal carcinomas. Anti-EGFR antibody (cetuximab) is considered to be a molecularly targeted agent for unresectable advanced colorectal carcinomas. The increase in incidence of right-sided colon carcinomas as well as the increase in the number of patients presenting with colorectal carcinomas means this issue should be addressed. Sessile serrated adenoma/polyp (SSA/P) with b-raf mutation and CIMP (CpG island methylator phenotype) abnormality as a precursor lesion of right-sided colon carcinoma is common and since cetuximab refractory wild-type K-ras/mutant b-raf colorectal carcinoma may increase in elderly patients and patients with right-sided colon carcinoma, a simultaneous examination for the K-ras and b-raf gene abnormalities for the treatment of colorectal cancer using anti-EGFR antibody (cetuximab) is crucial. In addition, the multidisciplinary assessments regarding the effect of such treatments is likely to be determined based on cumulative results, such as the duration of patient survival.
PMCID: PMC3915678  PMID: 24649163
unresectable advanced colorectal carcinomas; refractory to anti-epidermal growth factor receptor; cetuximab; epidermal growth factor receptor downstream pathway; K-ras gene; b-raf gene; right side colon
11.  Intrasynovial Tendon Graft for Chronic Flexor Tendon Laceration of the Finger: A Case Report 
We present the case of a patient with flexor digitorum profundus tendon laceration at the A2 pulley level caused by an injury to the base of the right ring finger by a knife. The patient was treated by flexor tendon reconstruction from the palm to the fingertip by using the left second toe flexor tendon as a graft, which improved the active range of motion. Further improvement was achieved by subsequent tenolysis, which eventually restored nearly normal function. Our experience with this case indicates that the intrasynovial tendon is a reasonable graft source for the synovial space in fingers and may enable restoration of excellent postoperative function.
PMCID: PMC3763753  PMID: 24015158
Flexor tendon graft; intrasynovial tendon; friction; tenolysis.
12.  Joint Pain Undergoes a Transition in Accordance with Signal Changes of Bones Detected by MRI in Hip Osteoarthritis 
In this study, we aimed to investigate whether joint pain is derived from cartilage or bone alterations.
We reviewed 23 hip joints of 21 patients with primary hip osteoarthritis (OA), which were classified into Kellgren–Laurence (KL) grading I to IV. Plain radiographs and magnetic resonance imaging (MRI) were obtained from all of the 23 joints. Two of the 21 patients had bilateral hip OA. Pain was assessed based on the pain scale of Denis. A Welch t test was performed for age, height, weight, body mass index, bone mineral density, and a Mann–Whitney U test was performed for KL grading.
Four of 8 hip joints with pain and OA showed broad signal changes detected by MRI. Fourteen hip joints without pain, but with OA did not show broad signal changes by MRI. Collectively, MRI analyses showed that broad signal changes in OA cases without joint pain or with a slight degree of joint pain were not observed, while broad signal changes were observed in OA cases with deteriorated joint pain.
Our findings suggest that hip joint pain might be associated with bone signal alterations in the hips of OA patients.
PMCID: PMC3795405  PMID: 24133552
Hip OA; MRI; signal changes; joint pain.
13.  The Pathophysiology of Primary Hip Osteoarthritis may Originate from Bone Alterations 
The aim of this study was to investigate whether bone alterations detected by hip magnetic resonance imaging (MRI) were associated with subsequent primary hip OA.
We enrolled 7 patients with hip joint pain from their first visit, at which hip joints were classified as grade 0 or I on the Kellgren-Lawrence grading scale. Plain radiographs and magnetic resonance imaging (MRI) were performed on all cases, and pain was assessed with the Denis pain scale. Average age, height, weight, body mass index, bone mineral density (L1-4), central edge angle, Sharp’s angle, and acetabular hip index were calculated.
Within two months of the onset of pain, 4 of the 7 cases showed broad bone signal changes, while 3 cases showed local signal changes in the proximal femur on hip MRI. Three to 6 months after the onset of pain, in all patients whose pain was much improved, plain radiographs showed progression to further-stage OA.
Our findings suggest that bone abnormalities in the proximal femur might be involved in the pathogenesis of primary hip OA.
PMCID: PMC3866704  PMID: 24358070
Pathophysiology; bone; hip osteoarthritis; MRI; joint pain.
14.  Cervical Pedicle Screw Fixation Combined with Laminoplasty for Cervical Spondylotic Myelopathy with Instability 
Asian Spine Journal  2012;6(4):241-248.
Study Design
A retrospective study.
To evaluate the surgical results of cervical pedicle screw (CPS) fixation combined with laminoplasty for treating cervical spondylotic myelopathy (CSM) with instability.
Overview of Literature
Cervical fixation and spinal cord decompression are required for CSM patients with instability. However, only a few studies have reported on CPS fixation combined with posterior decompression for unstable CSM patients.
Thirteen patients that underwent CPS fixation combined with laminoplasty for CSM with instability were evaluated in this study. We assessed the clinical and radiological results of the surgical procedures. The Japanese Orthopedic Association (JOA) scoring system was used to evaluate the clinical results. The percentages of sli p, difference in sli p angle between maximum flexion and maximum extension of unstable intervertebrae, and perforation rate of CPS were evaluated.
The mean JOA scores before surgery, immediately after surgery, and at final follow-up were 9.1, 13.3, and 12.6, respectively. The mean percentages of sli p before surgery, immediately after surgery, and at final follow-up were 9.1%, 3.2%, and 3.5%, respectively; there were significant improvements immediately after surgery and at final follow-up. The difference in sli p angle between the maximum flexion and maximum extension of the unstable intervertebrae changed from 9.0° before surgery to 1.6° at the final follow-up. The perforation rate of CPS was 10.9%.
The results suggest that CPS fixation combined with laminoplasty is an effective surgical procedure for treating CSM with instability.
PMCID: PMC3530698  PMID: 23275807
Cervical spondylosis; Myelopathy; Instability; Cervical fixation
15.  A Case of Pyogenic Spondylodiscitis Caused by Campylobacter fetus for Which Early Diagnosis by Magnetic Resonance Imaging Was Difficult 
Asian Spine Journal  2012;6(4):274-278.
The purpose of this case report was to report a rare case of pyogenic spondylodiscitis caused by Campylobacter fetus. A 37-year-old male presented with fever and low back pain. By lumbar magnetic resonance imaging (MRI), no abnormal finding was observed at the first presentation. However, low back pain was aggravated, and fever did not improve. Thus, lumbar MRI was repeated on the 26 day after the onset of symptoms, showing abnormal signals at vertebrae and disc spaces, and pyogenic spondylitis was diagnosed. The possibility of pyogenic spondylodiscitis should be taken into account if a patient presents with low back pain and fever, and areas of low signal intensity on a T1-weighted MRI should be carefully examined. When initial MRI does not reveal abnormal findings, repeated MRI after one or two weeks or, more favorably, immediate gadolinium enhancement MRI, are important for patients who have persistent low back pain and fever.
PMCID: PMC3530702  PMID: 23275811
Resonance imaging; Campylobacter fetus
16.  Baseline Serum Cholesterol Is Associated with a Response to Pegylated Interferon Alfa-2b and Ribavirin Therapy for Chronic Hepatitis C Genotype 2 
Background. HCV infection is associated with lipid disorders because this virus utilizes the host lipid metabolism to sustain its life cycle. Several studies have indicated that higher concentrations of serum cholesterol and LDL before treatment are important predictors of higher rates of sustained virological response (SVR). However, most of these studies involved patients infected with HCV genotype 1. Thus, we performed a multi-institutional clinical study to evaluate the impact of lipid profiles on SVR rates in patients with HCV genotype 2. Methods. A total of 100 chronic hepatitis C patients with HCV genotype 2 who received peg-IFN alfa-2b and ribavirin therapy were consecutively enrolled. The significance of age, sex, BMI, AST level, ALT level, WBC, hemoglobin, platelet count, gamma-glutamyltransferase, total cholesterol level (TC), LDL level, HCV RNA, and histological evaluation was examined for SVR using logistic regression analysis. Results. The 100 patients infected with HCV genotype 2 were divided into 2 groups, an SVR group and a non-SVR group. Characteristics of each group were subsequently compared. There was no significant difference in the level of HCV RNA, BMI, platelet, TG, or stage of fibrosis between the groups. However, there were significant differences in the levels of TC and LDL-C. In multivariate logistic regression analysis using baseline characteristics, high TC level was an independent and significant risk factor (relative risk 18.59, P = 0.015) for SVR. Conclusion. Baseline serum total cholesterol levels should be considered when assessing the likelihood of sustained treatment response following the course of peg-IFN and ribavirin therapy in patients with chronic HCV genotype 2 infection.
PMCID: PMC3501951  PMID: 23193392
17.  Differentiation between sessile serrated adenoma/polyp and non-sessile serrated adenoma/polyp in large hyper plastic polyp: A Japanese collaborative study 
A hyperplastic polyp (HP) >10 mm is described as a large hyperplastic polyp (LHP). Previous studies have considered LHP and sessile serrated adenoma/polyp (SSA/P) as synonymous. Although HP and SSA/P have previously been morphologically distinguished, differences between LHP and SSA/P have not yet been reported. The present study aimed to define the differences between SSA/P and non-SSA/P in LHP using immunohistochemistry for Ki67. Colorectal serrated lesions (>10 mm) that were completely resected by endoscope and derived from 11 institutions in Japan [Dokkyo Medical University School of Medicine (Mibu), Takahiro Fujii Clinic (Tokyo), Sano Hospital (Kobe), Oda GI Clinic, Hattori GI Endoscopy and Oncology Clinic (Kumamoto), Ohta Clinic (Nagoya), Hiroshima University (Hiroshima), Iwate Medical University (Morioka), Juntendo and Kyorin Universities (Tokyo) as well as Toyama University (Toyama)] affiliated with the Japanese Society for Cancer of the Colon and Rectum (JSCCR) between January 2003 and December 2010 were selected. The histological criteria of the Japanese Society for Cancer of the Colon and Rectum (JSCCR, project meeting; editor-in chief, Takashi Yao) were used to distinguish SSA/P and non-SSA/P from LHP. Non-SSA/P comprises both incomplete SSA/P and HP. A total of 154 samples diagnosed as SSA/P or non-SSA/P from 148 patients were used. This study comprised 107 SSA/P and 47 non-SSA/P cases, whereby lesions were located on the right side of the colon (73.2 and 26.8%, respectively). Ki67-positivity in SSA/Ps was significantly higher compared to non-SSA/Ps. A greater number of SSA/Ps in LHP were located on the right side of the colon compared to the left side. SSA/Ps occurring on the right side of the colon may be precursor lesions of colorectal carcinoma in serrated neoplasia pathways. In conclusion, LHPs and SSA/Ps limited to the right side of the colon are suggested to be clinically treated as the same type of lesions.
PMCID: PMC3956251  PMID: 24649122
large hyperplastic polyp; sessile serrated adenoma/polyp; Ki67; tumor location
18.  Computer-assisted C1-C2 Transarticular Screw Fixation "Magerl Technique" for Atlantoaxial Instability 
Asian Spine Journal  2012;6(3):168-177.
Study Design
A retrospective study.
To evaluate the surgical results of computer-assisted C1-C2 transarticular screw fixation for atlantoaxial instability and the usefulness of the navigation system.
Overview of Literature
We used a computed tomography (CT)-based computer navigation system in planning and screw insertion in Magerl's procedure, which provides the most rigid atlantoaxial fusion, to avoid risk of vertebral artery (VA) tear by avoiding high-riding VA during screw insertion.
Twenty patients who underwent atlantoaxial fusion under the CT-based navigation system were studied. The mean observation period was 33.5 months. The evaluated items included the existence of VA stenosis by preoperative magnetic resonance angiography, surgical time, blood loss volume, Japanese Orthopaedic Association (JOA) score and Ranawat's pain criteria before surgery and at final follow-up, postoperative screw position evaluated by CT, and bony fusion.
The mean operation time was 205 minutes, with the mean blood loss volume of 242 ml. The mean JOA score was 11.6 points before surgery and 13.7 at final follow-up. Occipital and/or cervical pain presented before operation was remitted or resolved in all patients. Evaluation of screw insertion by CT revealed correct penetration to atlantoaxial joints, with a perforation rate of 2.6%. There was no complication, including VA tear, and all patients who were followed-up during one year or more after surgery achieved bony fusion. Some subjects who appeared inappropriate for surgery from CT images were assessed as eligible for surgery based on the evaluation results obtained using the navigation system.
It was demonstrated that the CT-based navigation system is an effective support device for Magerl's procedure.
PMCID: PMC3429607  PMID: 22977696
Atlantoaxial joint; Atlantoaxial instability; CT-based computer navigation system; C1-C2 transarticular screw fixation
19.  Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice 
Scientific Reports  2012;2:498.
The application of carbon nanotubes (CNTs) as biomaterials is of wide interest, and studies examining their application in medicine have had considerable significance. Biological safety is the most important factor when considering the clinical application of CNTs as biomaterials, and various toxicity evaluations are required. Among these evaluations, carcinogenicity should be examined with the highest priority; however, no report using transgenic mice to evaluate the carcinogenicity of CNTs has been published to date. Here, we performed a carcinogenicity test by implanting multi-walled CNTs (MWCNTs) into the subcutaneous tissue of rasH2 mice, using the carbon black present in black tattoo ink as a reference material for safety. The rasH2 mice did not develop neoplasms after being injected with MWCNTs; instead, MWCNTs showed lower carcinogenicity than carbon black. Such evaluations should facilitate the clinical application and development of CNTs for use in important medical fields.
PMCID: PMC3391660  PMID: 22787556
20.  Histone deacetylase-4 is required during early cranial neural crest development for generation of the zebrafish palatal skeleton 
Histone deacetylase-4 (Hdac4) is a class II histone deacetylase that inhibits the activity of transcription factors. In humans, HDAC4 deficiency is associated with non-syndromic oral clefts and brachydactyly mental retardation syndrome (BDMR) with craniofacial abnormalities.
We identify hdac4 in zebrafish and characterize its function in craniofacial morphogenesis. The gene is present as a single copy, and the deduced Hdac4 protein sequence shares all known functional domains with human HDAC4. The zebrafish hdac4 transcript is widely present in migratory cranial neural crest (CNC) cells of the embryo, including populations migrating around the eye, which previously have been shown to contribute to the formation of the palatal skeleton of the early larva. Embryos injected with hdac4 morpholinos (MO) have reduced or absent CNC populations that normally migrate medial to the eye. CNC-derived palatal precursor cells do not recover at the post-migratory stage, and subsequently we found that defects in the developing cartilaginous palatal skeleton correlate with reduction or absence of early CNC cells. Palatal skeletal defects prominently include a shortened, clefted, or missing ethmoid plate, and are associated with a shortening of the face of young larvae.
Our results demonstrate that Hdac4 is a regulator of CNC-derived palatal skeletal precursors during early embryogenesis. Cleft palate resulting from HDAC4 mutations in human patients may result from defects in a homologous CNC progenitor cell population.
PMCID: PMC3426487  PMID: 22676467
Cartilage; hdac4; Neural crest; Palate; Skeleton; Zebrafish
21.  Elucidation mechanism of different biological responses to multi-walled carbon nanotubes using four cell lines 
We examined differences in cellular responses to multi-walled carbon nanotubes (MWCNTs) using malignant pleural mesothelioma cells (MESO-1), bronchial epithelial cells (BEAS-2B), neuroblastoma cells (IMR-32), and monoblastic cells (THP-1), before and after differentiation. MESO-1, BEAS-2B and differentiated THP-1 cells actively endocytosed MWCNTs, resulting in cytotoxicity with lysosomal injury. However, cytotoxicity did not occur in IMR-32 or undifferentiated THP-1 cells. Both differentiated and undifferentiated THP-1 cells exhibited an inflammatory response. Carbon blacks were endocytosed by the same cell types without lysosomal damage and caused cytokine secretion, but they did not cause cytotoxicity. These results indicate that the cytotoxicity of MWCNTs requires not only cellular uptake but also lysosomal injury. Furthermore, it seems that membrane permeability or cytokine secretion without cytotoxicity results from several active mechanisms. Clarification of the cellular recognition mechanism for MWCNTs is important for developing safer MWCNTs.
PMCID: PMC3260041  PMID: 22267932
multi-walled carbon nanotubes; cytotoxicity; endocytosis; cytokine secretion; reactive oxygen species
22.  Effect of dispersants of multi-walled carbon nanotubes on cellular uptake and biological responses 
Although there have been many reports about the cytotoxicity of multi-walled carbon nanotubes (MWCNTs), the results are still controversial. To investigate one possible reason, the authors investigated the influence of MWCNT dispersants on cellular uptake and cytotoxicity. Cytotoxicity was examined (measured by alamarBlue® assay), as well as intracellular MWCNT concentration and cytokine secretion (measured by flow cytometry) in human bronchial epithelial cells (BEAS-2B) exposed to a type of highly purified MWCNT vapor grown carbon fiber (VGCF®, Shōwa Denkō Kabushiki-gaisha, Tokyo, Japan) in three different dispersants (gelatin, carboxylmethyl cellulose, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The authors also researched the relationship between the intracellular concentration of MWCNTs and cytotoxicity by using two cell lines, BEAS-2B and MESO-1 human malignant pleural mesothelioma cells. The intracellular concentration of VGCF was different for each of the three dispersants, and the levels of cytotoxicity and inflammatory response were correlated with the intracellular concentration of VGCF. A relationship between the intracellular concentration of VGCF and cytotoxic effects was observed in both cell lines. The results indicate that dispersants affect VGCF uptake into cells and that cytotoxicity depends on the intracellular concentration of VGCF, not on the exposed dosage. Thus, toxicity appears to depend on exposure time, even at low VGCF concentrations, because VGCF is biopersistent.
PMCID: PMC3252677  PMID: 22228997
multi-walled carbon nanotube; cytotoxicity; intracellular concentration; dispersant; cytokine secretion
23.  DJ-1 as a potential biomarker for the development of biocompatible multiwalled carbon nanotubes 
In the present study, we investigated whether DJ-1 could serve as a biomarker for assessing the biocompatibility of multiwalled carbon nanotubes (MWCNTs), using the highly purified carbon nanotube, HTT2800.
Using Western blot analysis, we determined DJ-1 protein levels in two different types of cells (one capable and the other incapable of HTT2800 endocytosis). Using quantitative real-time polymerase chain reaction, we also investigated the ability of purified nanotubes to alter DJ-1 mRNA levels.
We demonstrated that the DJ-1 protein concentration was reduced, regardless of the cytotoxic activity of intracellular HTT2800. Furthermore, HTT2800 decreased the DJ-1 mRNA levels in a dose-dependent manner. This decrease in DJ-1 mRNA levels was not observed in the case of Sumi black or cup-stacked carbon nanotubes.
These data indicate that modification of DJ-1 expression is caused by the cell response to MWCNTs. We conclude that DJ-1 is a promising candidate biomarker for the development of biocompatible MWCNTs.
PMCID: PMC3218582  PMID: 22114499
multiwalled carbon nanotubes; DJ-1 protein; Western blot; quantitative real-time polymerase chain reaction
24.  Esophageal granular cell tumor successfully resected by endoscopic submucosal dissection 
Esophagus  2011;8(3):203-207.
Granular cell tumors of the esophagus are rare neoplasms and their diagnosis is mainly based on histopathologic examination of endoscopic biopsies. With the development of endoscopic techniques, there has been a marked increase in local treatment modalities for early esophageal neoplasms. In this case report, we describe the removal of a granular cell tumor by the endoscopic submucosal dissection technique, and briefly discuss the literature on clinicopathologic aspects and management of granular cell tumors.
PMCID: PMC3339600  PMID: 22593727
Biopsy; Esophageal neoplasms; Granular cell tumor; Surgical endoscopy; Medicine & Public Health; Oncology; Thoracic Surgery; Pathology; Gastroenterology; Surgical Oncology
25.  Early Changes in Bone Specific Turnover Markers During the Healing Process After Vertebral Fracture 
The present study measured longitudinal changes in bone turnover markers in elderly patients with vertebral fracture and investigated the relationship among bone turnover markers, duration of bed rest and bone mineral density (BMD).
Criteria for patient selection were 50 years in age and older, and presence of VF. Serum bone-specific alkaline phosphatase (BAP) was measured as a marker of bone formation. Urinary crosslinked N-terminal telopeptides of type I collagen (NTX) was measured as a marker of bone resorption. In principle, samples were collected just after injury, within 24 h, and 1, 2, 3, 5 and 8 weeks after. We also measured duration of bed rest and BMD.
The study population consisted of 42 cases. The average BMD of the lumbar vertebrae was 0.670 ± 0.174 g/cm2. Bed rest period was 17.9 ± 8.8 days. BAP showed significantly higher values at 2 and 3 weeks compared with the baseline value. Thereafter, BAP progressively decreased until 8 weeks. Urinary NTX was increased soon after the onset of pain with the same patterns in BAP. Urinary NTX values reached a peak at 3 weeks, and then they kept significantly higher values until 8 weeks. The peak value of serum BAP was affected by the duration of bed rest, although that of the urinary NTX was not. The peak values of serum BAP and urinary NTX showed negative correlations with the initial BMD values.
Bone turnover markers remained higher at 8 weeks, even patients symptom was healed after VF. Bone turnover markers were affected on physical activity and BMD.
PMCID: PMC3087216  PMID: 21552459
Vertebral fracture; bone turnover marker; osteoporosis.

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