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1.  Antiangiogenic and Antitumor Activities of Aflibercept, a Soluble VEGF Receptor-1 and -2, in a Mouse Model of Hepatocellular Carcinoma 
Neoplasia (New York, N.Y.)  2016;18(7):413-424.
BACKGROUND & AIM: Aflibercept known as ziv-aflibercept in the United States is a soluble decoy receptor of both vascular endothelial growth factor (VEGF) receptor-1 and -2 known to inhibit the binding of VEGF and placental growth factor (PlGF) to VEGF receptor-1 and -2. Here, we analyzed the mechanisms of the antitumor effects of aflibercept in mouse hepatoma models. METHODS: In in vitro studies, we determined the effects of aflibercept on human umbilical vein cell (HUVEC) proliferation and bone marrow (BM) cell differentiation to endothelial progenitor cells (EPCs). In in vivo experiments, aflibercept was injected intraperitoneally in hepatoma cell tumor-bearing mice, and its inhibitory effects on tumor growth and BM cell migration to tumor tissues were evaluated. RESULTS: Aflibercept suppressed phosphorylation of VEGF receptor-1 and -2 in HUVEC and dose-dependently inhibited VEGF-induced HUVEC proliferation. It suppressed the differentiation of BM cells to EPCs and migration of BM cells to tumor tissues. It also suppressed tumor growth and prolonged survival time of tumor-bearing mice without side effects. In tumor tissues, aflibercept upregulated the expression of hypoxia inducible factor1-α, VEGF, PlGF, fibroblast growth factor-2, platelet derived growth factor-BB, and transforming growth factor-α and reduced microvascular density. It also reduced sinusoidal density in noncancerous liver tissues. CONCLUSIONS: Our results demonstrated potent antitumor activity for aflibercept in a mouse model of hepatocellular carcinoma. These effects were mediated through inhibition of neovascularization, caused by inhibition of endothelial cell proliferation, EPC differentiation, and BM cell migration to tumor tissues.
PMCID: PMC4954942  PMID: 27435924
2.  Somatic mutations in GLI3 and OFD1 involved in sonic hedgehog signaling cause hypothalamic hamartoma 
Hypothalamic hamartoma (HH) is a congenital anomalous brain tumor. Although most HHs are found without any other systemic features, HH is observed in syndromic disorders such as Pallister–Hall syndrome (PHS) and oral‐facial‐digital syndrome (OFD). Here, we explore the possible involvement of somatic mutations in HH.
We analyzed paired blood and hamartoma samples from 18 individuals, including three with digital anomalies, by whole‐exome sequencing. Detected somatic mutations were validated by Sanger sequencing and deep sequencing of target amplicons. The effect of GLI3 mutations on its transcriptional properties was evaluated by luciferase assays using reporters containing eight copies of the GLI‐binding site and a mutated control sequence disrupting GLI binding.
We found hamartoma‐specific somatic truncation mutations in GLI3 and OFD1, known regulators of sonic hedgehog (Shh) signaling, in two and three individuals, respectively. Deep sequencing of amplicons covering the mutations showed mutant allele rates of 7–54%. Somatic mutations in OFD1 at Xp22 were found only in male individuals. Potential pathogenic somatic mutations in UBR5 and ZNF263 were also identified in each individual. Germline nonsense mutations in GLI3 and OFD1 were identified in each individual with PHS and OFD type I in our series, respectively. The truncated GLI3 showed stronger repressor activity than the wild‐type protein. We did not detect somatic mutations in the remaining 9 individuals.
Our data indicate that a spectrum of human disorders can be caused by lesion‐specific somatic mutations, and suggest that impaired Shh signaling is one of the pathomechanisms of HH.
PMCID: PMC4863748  PMID: 27231705
3.  Spatiotemporal Accuracy of Gradient Magnetic-Field Topography (GMFT) Confirmed by Simultaneous Magnetoencephalography and Intracranial Electroencephalography Recordings in Patients with Intractable Epilepsy 
Gradient magnetic-field topography (GMFT) is one method for analyzing magnetoencephalography (MEG) and representing the spatiotemporal dynamics of activity on the brain surface. In contrast to spatial filters, GMFT does not include a process reconstructing sources by mixing sensor signals with adequate weighting. Consequently, noisy sensors have localized and limited effects on the results, and GMFT can handle MEG recordings with low signal-to-noise ratio. This property is derived from the principle of the planar-type gradiometer, which obtains maximum gradient magnetic-field signals just above the electrical current source. We assumed that this characteristic allows GMFT to represent even faint changes in brain activities that cannot be achieved with conventional equivalent current dipole analysis or spatial filters. GMFT is thus hypothesized to represent brain surface activities from onset to propagation of epileptic discharges. This study aimed to validate the spatiotemporal accuracy of GMFT by analyzing epileptic activities using simultaneous MEG and intracranial electroencephalography (iEEG) recordings. Participants in this study comprised 12 patients with intractable epilepsy. Epileptic spikes simultaneously detected on both MEG and iEEG were analyzed by GMFT and voltage topography (VT), respectively. Discrepancies in spatial distribution between GMFT and VT were evaluated for each epileptic spike. On the lateral cortices, areas of GMFT activity onset were almost concordant with VT activities arising at the gyral unit level (concordance rate, 66.7–100%). Median time lag between GMFT and VT at onset in each patient was 11.0–42.0 ms. On the temporal base, VT represented basal activities, whereas GMFT failed but instead represented propagated activities of the lateral temporal cortices. Activities limited to within the basal temporal or deep brain region were not reflected on GMFT. In conclusion, GMFT appears to accurately represent brain activities of the lateral cortices at the gyral unit level. The slight time lag between GMFT and VT is likely attributable to differences in the detection principles underlying MEG and iEEG. GMFT has great potential for investigating the spatiotemporal dynamics of lateral brain surface activities.
PMCID: PMC4990550  PMID: 27594827
magnetoencephalography; gradient magnetic-field topography; spatiotemporal resolution; simultaneous recording; intracranial electroencephalography; voltage topography
4.  Long-term outcomes of rotational atherectomy in coronary bifurcation lesions 
The aim of the present study was to determine the long-term outcomes of bifurcation lesions following a rotational atherectomy (ROTA). In this retrospective study, patients that had undergone a ROTA of the bifurcation coronary lesions in Juntendo University Hospital (Tokyo, Japan) were enrolled between January 2007 and December 2009, and received follow-up for a median duration of 48 months (range, 12–84 months). A total of 337 patients were enrolled. Each patient was treated with an average of 1.2±0.4 ROTA burrs (mean size, 2.9±0.3 mm). Baseline lesion length, reference diameter, minimal lumen diameter (MLD) and percentage of diameter stenosis (%DS) prior to the procedure were comparable between the DM and non-DM patients. Furthermore, MLD, %DS and acute gain following the procedure were similar between the two groups. At follow-up, DM patients exhibited a significantly decreased MLD (1.97±0.92 vs. 2.26±0.73 mm; P=0.0038), increased %DS (27.9±21.3 vs. 20.2±13.3%; P=0.022) and late loss (0.70±0.45 vs. 0.42±0.36 mm; P=0.0047) compared with the non-DM patients. Follow-up examinations (mean duration, 52.2±19.4 months) revealed that the DM patients experienced significantly higher rates of target lesion revascularization (TLR) [28 (15.7%) vs. 8 (5.0%); P=0.0011], target lesion (TL) restenosis [46 (25.8%) vs. 20 (12.6%); P=0.0019] and major adverse cardiac events (MACE) [36 (20.2%) vs. 19 (12.0%), P=0.039] compared with the non-DM patients. Although the usage of ROTA and drug-eluting stent evidently improved long-term outcomes in patients with bifurcation lesions, DM remained an independent risk factor for TLR, TL restenosis and MACE. Therefore, the management of DM in bifurcation lesions treated with ROTA requires increased investigation in future clinical practice.
PMCID: PMC4665993  PMID: 26668644
rotational atherectomy; long-term outcomes; coronary bifurcation lesion
5.  Iron biofortification of rice using different transgenic approaches 
Rice  2013;6(1):40.
More than 2 billion people suffer from iron (Fe) deficiency, and developing crop cultivars with an increased concentration of micronutrients (biofortification) can address this problem. In this review, we describe seven transgenic approaches, and combinations thereof, that can be used to increase the concentration of Fe in rice seeds. The first approach is to enhance the Fe storage capacity of grains through expression of the Fe storage protein ferritin under the control of endosperm-specific promoters. Using this approach, the concentration of Fe in the seeds of transformants was increased by approximately 2-fold in polished seeds. The second approach is to enhance Fe translocation by overproducing the natural metal chelator nicotianamine; using this approach, the Fe concentration was increased by up to 3-fold in polished seeds. The third approach is to enhance Fe influx to the endosperm by expressing the Fe(II)-nicotianamine transporter gene OsYSL2 under the control of an endosperm-specific promoter and sucrose transporter promoter, which increased the Fe concentration by up to 4-fold in polished seeds. The fourth approach is introduction of the barley mugineic acid synthesis gene IDS3 to enhance Fe uptake and translocation within plants, which resulted in a 1.4-fold increase in the Fe concentration in polished seeds during field cultivation. In addition to the above approaches, Fe-biofortified rice was produced using a combination of the first, second, and third approaches. The Fe concentration in greenhouse-grown T2 polished seeds was 6-fold higher and that in paddy field-grown T3 polished seeds was 4.4-fold higher than in non-transgenic seeds without any reduction in yield. When the first and fourth approaches were combined, the Fe concentration was greater than that achieved by introducing only the ferritin gene, and Fe-deficiency tolerance was observed. With respect to Fe biofortification, the introduction of multiple Fe homeostasis genes is more effective than the introduction of individual genes. Moreover, three additional approaches, i.e., overexpression of the Fe transporter gene OsIRT1 or OsYSL15, overexpression of the Fe deficiency-inducible bHLH transcription factor OsIRO2, and knockdown of the vacuolar Fe transporter gene OsVIT1 or OsVIT2, may be useful to further increase the Fe concentration of seeds.
PMCID: PMC3878263  PMID: 24351075
Biofortification; Iron; Zinc; Transgenic rice; Nicotianamine; YSL; Ferritin; IDS3; Mugineic acids; Anemia
6.  Iron-biofortification in rice by the introduction of three barley genes participated in mugineic acid biosynthesis with soybean ferritin gene 
Iron deficiency is a serious problem around the world, especially in developing countries. The production of iron-biofortified rice will help ameliorate this problem. Previously, expression of the iron storage protein, ferritin, in rice using an endosperm-specific promoter resulted in a two-fold increase in iron concentration in the resultant transgenic seeds. However, further over expression of ferritin did not produce an additional increase in the seed iron concentration, and symptoms of iron deficiency were noted in the leaves of the transgenic plants. In the present study, we aimed to further increase the iron concentration in rice seeds without increasing the sensitivity to iron deficiency by enhancing the uptake and transport of iron via a ferric iron chelator, mugineic acid. To this end, we introduced the soybean ferritin gene (SoyferH2) driven by two endosperm-specific promoters, along with the barley nicotianamine synthase gene (HvNAS1), two nicotianamine aminotransferase genes (HvNAAT-A and -B), and a mugineic acid synthase gene (IDS3) to enhance mugineic acid production in rice plants. A marker-free vector was utilized as a means of increasing public acceptance. Representative lines were selected from 102 transformants based on the iron concentration in polished seeds and ferritin accumulation in the seeds. These lines were grown in both commercially supplied soil (iron-sufficient conditions) and calcareous soil (iron-deficient conditions). Lines expressing both ferritin and mugineic acid biosynthetic genes showed signs of iron-deficiency tolerance in calcareous soil. The iron concentration in polished T3 seeds was increased by 4 and 2.5 times, as compared to that in non-transgenic lines grown in normal and calcareous soil, respectively. These results indicate that the concomitant introduction of the ferritin gene and mugineic acid biosynthetic genes effectively increased the seed iron level without causing iron sensitivity under iron-limited conditions.
PMCID: PMC3653162  PMID: 23675379
anemia; iron; zinc; rice; mugineic acid; biofortification; ferritin; IDS3
7.  Iron Biofortification of Myanmar Rice 
Iron (Fe) deficiency elevates human mortality rates, especially in developing countries. In Myanmar, the prevalence of Fe-deficient anemia in children and pregnant women are 75 and 71%, respectively. Myanmar people have one of the highest per capita rice consumption rates globally. Consequently, production of Fe-biofortified rice would likely contribute to solving the Fe-deficiency problem in this human population. To produce Fe-biofortified Myanmar rice by transgenic methods, we first analyzed callus induction and regeneration efficiencies in 15 varieties that are presently popular because of their high-yields or high-qualities. Callus formation and regeneration efficiency in each variety was strongly influenced by types of culture media containing a range of 2,4-dichlorophenoxyacetic acid concentrations. The Paw San Yin variety, which has a high-Fe content in polished seeds, performed well in callus induction and regeneration trials. Thus, we transformed this variety using a gene expression cassette that enhanced Fe transport within rice plants through overexpression of the nicotianamine synthase gene HvNAS1, Fe flow to the endosperm through the Fe(II)-nicotianamine transporter gene OsYSL2, and Fe accumulation in endosperm by the Fe storage protein gene SoyferH2. A line with a transgene insertion was successfully obtained. Enhanced expressions of the introduced genes OsYSL2, HvNAS1, and SoyferH2 occurred in immature T2 seeds. The transformants accumulated 3.4-fold higher Fe concentrations, and also 1.3-fold higher zinc concentrations in T2 polished seeds compared to levels in non-transgenic rice. This Fe-biofortified rice has the potential to reduce Fe-deficiency anemia in millions of Myanmar people without changing food habits and without introducing additional costs.
PMCID: PMC3664312  PMID: 23750162
iron; anemia; biofortification; nicotianamine; ferritin; OsYSL2; Myanmar rice; rice transformation
8.  Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition 
Scientific Reports  2012;2:543.
To address the problem of iron-deficiency anemia, one of the most prevalent human micronutrient deficiencies globally, iron-biofortified rice was produced using three transgenic approaches: by enhancing iron storage in grains via expression of the iron storage protein ferritin using endosperm-specific promoters, enhancing iron translocation through overproduction of the natural metal chelator nicotianamine, and enhancing iron flux into the endosperm by means of iron(II)-nicotianamine transporter OsYSL2 expression under the control of an endosperm-specific promoter and sucrose transporter promoter. Our results indicate that the iron concentration in greenhouse-grown T2 polished seeds was sixfold higher and that in paddy field-grown T3 polished seeds was 4.4-fold higher than that in non-transgenic seeds, with no defect in yield. Moreover, the transgenic seeds accumulated zinc up to 1.6-times in the field. Our results demonstrate that introduction of multiple iron homeostasis genes is more effective for iron biofortification than the single introduction of individual genes.
PMCID: PMC3408131  PMID: 22848789
9.  Strategies for Regenerating Striatal Neurons in the Adult Brain by Using Endogenous Neural Stem Cells 
Currently, there is no effective treatment for the marked neuronal loss caused by neurodegenerative diseases, such as Huntington's disease (HD) or ischemic stroke. However, recent studies have shown that new neurons are continuously generated by endogenous neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain, including the human brain. Because some of these new neurons migrate to the injured striatum and differentiate into mature neurons, such new neurons may be able to replace degenerated neurons and improve or repair neurological deficits. To establish a neuroregenerative therapy using this endogenous system, endogenous regulatory mechanisms that can be co-opted for efficient regenerative interventions must be understood, along with any potential drawbacks. Here, we review current knowledge on the generation of new neurons in the adult brain and discuss their potential for use in replacing striatal neurons lost to neurodegenerative diseases, including HD, and to ischemic stroke.
PMCID: PMC3135217  PMID: 21766028
10.  Surgical correction of buried penis after traffic accident – a case report 
BMC Urology  2004;4:6.
Buried penis, most commonly seen in children, is particularly debilitating in adults, resulting in inability to void while standing and it also affects vaginal penetration. We report a case of buried penis due to a traffic accident, which caused dislocation of the fractured pubic bone that shifted inside and pulled the penis by its suspensory ligament.
Case presentation
A 55-year-old man was admitted to our hospital with a chief complaint of hidden penis while in the sitting position. He had suffered a pelvic fracture in a traffic accident four years previously, and his penis was covered with suprapubic fat when he was in a sitting position. He was unable to have sexual intercourse. We performed a penile lengthening procedure, including inverse V-Y-plasty of the dorsal skin of the penile root, suspensory desmotomy and fat removal, under general anesthesia. There was a good cosmetic result with satisfactory penile erection, which allowed successful sexual intercourse after surgery.
We performed penile elongation surgery with inverse V-Y-plasty of the dorsal skin of the penile root, suspensory desmotomy, and fat removal. Surgical treatment of buried penis achieves marked aesthetic and functional improvement, and benefits the majority of patients, resulting in satisfactory erection and successful sexual intercourse.
PMCID: PMC434514  PMID: 15182380
pelvic fracture; buried penis; V-Yplasty
11.  Adult Wilms' tumor with calcification untreated for 5 years – a case report 
BMC Urology  2004;4:5.
Wilms' tumor is rarely found in adults and there are no established treatment guidelines for such tumors in adults. Whereas calcification is a common finding in neuroblastoma, it is considered uncommon in Wilms' tumor.
Case presentation
We report a case of adult Wilms' tumor with calcification in a 22-year-old man. He had been initially referred to our hospital with a chief complaint of right flank pain 5 years previously, when abdominal computed tomography had revealed focal calcification at the upper pole of the right kidney. Although we planned further assessment, he did not revisit our hospital again until 5 years later, again because of right flank pain. Ultrasound and computed tomography scan revealed a large mass lesion with calcification in the right kidney, invasive to the hepatic lobe. The patient underwent curative right nephrectomy and partial right hepatic lobectomy. Pathological analysis demonstrated a nephroblastoma (Wilms' tumor) with predominant epithelial histology infiltrating the hepatic lobe. The patient has been well without tumor recurrence for 15 months after surgery.
Calcification may be a sign of slow tumor gowth and possibly a favorable prognosis in cases of adult Wilms' tumor.
PMCID: PMC425587  PMID: 15180902
prognosis; nephroblastoma
12.  Accelerated intimal hyperplasia and increased endogenous inhibitors for NO synthesis in rabbits with alloxan-induced hyperglycaemia 
British Journal of Pharmacology  1999;126(1):211-218.
We examined whether endogenous inhibitors of NO synthesis are involved in the augmentation of intimal hyperplasia in rabbits with hyperglycaemia induced by alloxan.Four weeks after the endothelial denudation of carotid artery which had been performed 12 weeks after alloxan, the intimal hyperplasia was greatly augmented with hyperglycaemia. The degree of hyperplasia was assessed using three different parameters of histopathological findings as well as changes in luminal area and intima : media ratio.There were positive and significant correlations between intima : media ratio, plasma glucose, and concentrations of NG-monomethyl-L-arginine (L-NMMA) and NG, NG-dimethyl-L-arginine (ADMA) in endothelial cells, that is, the intima : media ratio became greater as plasma glucose and endothelial L-NMMA and ADMA were increased. Furthermore, endothelial L-NMMA and ADMA were increased in proportion to the increase in plasma glucose.In contrast, there were inverse and significant correlations between cyclic GMP production by carotid artery strips with endothelium and plasma glucose, between cyclic GMP production and endothelial L-NMMA and ADMA, and between the intima : media ratio and cyclic GMP production.Exogenously applied L-NMMA and ADMA inhibited cyclic GMP production in a concentration-dependent manner. IC50 values were determined to be 12.1 μM for the former and 26.2 μM for the latter. The cyclic GMP production was abolished after the deliberate removal of endothelium from the artery strips.These results suggest that the augmentation of intimal hyperplasia with hyperglycaemia is closely related to increased accumulation of L-NMMA and ADMA with hyperglycaemia, which would result in an accelerated reduction in NO production/release by endothelial cells.
PMCID: PMC1565802  PMID: 10051138
Hyperglycaemia; alloxan; accelerated intimal hyperplasia; L-NMMA; ADMA; NO production; endothelial denudation
13.  Endogenous asymmetrical dimethylarginine and hypertension associated with puromycin nephrosis in the rat 
British Journal of Pharmacology  1998;125(3):469-476.
The present experiments were designed to investigate the role of asymmetrical NG,NG-dimethyl-L-arginine (ADMA) in causing hypertension associated with the focal and segmental glomerulosclerosis (FSGS) produced by a single bolus of puromycin aminonucleoside (PAN) and successive injection of protamine for 7 days in rats which had undergone unilateral nephrectomy.After the unilateral nephrectomy, and administering PAN and protamine, histological examinations of the kidney revealed a typical FSGS, that is, evident abnormalities including segmental mesangial proliferation, obliteration of glomerular capillary lumens and adhesions between the glomerulus and Bowman's capsule could be observed. Changes in the glomerular epithelial cells consisted of the swelling with bleb formation.In the FSGS rats, urine volume and urinary protein were significantly (P<0.05 and P<0.005) increased throughout 4-week experimental period, while the creatinine clearance was significantly (P<0.005) and transiently decreased, and recovered 4 weeks later. These changes were associated with the sustained elevation of the systolic blood pressure.ADMA levels in aortic endothelial cells, plasma and urine were significantly (P<0.05 and P<0.005) increased in the FSGS rats, but the level in the kidney remained unchanged.The basal level and net production of cyclic GMP in the aortic vessel wall with endothelium when stimulated by norepinephrine and acetylcholine were significantly (P<0.05 and P<0.01) attenuated in the FSGS rats.There were significant and positive correlations between systolic blood pressure (y) and ADMA levels (x) in endothelial cells (y=4.43x+122.2, r=0.979, P<0.0001), plasma (y=0.10x+71.9, r=0.921, P<0.001) and urine (y=0.48x+126.9, r=0.699, P<0.005), but not significant in the kidney (y=0.06x+102.7, r=0.252, NS).These findings suggest that ADMA as an endogenous inhibitor of NO synthesis may play an important role for the pathogenesis in the hypertension associated with the experimental FSGS in the rat.
PMCID: PMC1565650  PMID: 9806329
ADMA; endogenous NOS inhibitor; FSGS; hypertension; puromycin

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