Publications by authors named "Takuya Nakayama"

34 Publications

Comparison between biodegradable- and durable-polymer everolimus-eluting stents in hemodialysis patients with coronary artery disease.

Cardiovasc Interv Ther 2021 Nov 24. Epub 2021 Nov 24.

Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

To investigate the clinical outcomes after biodegradable-polymer (BP) and durable-polymer (DP) everolimus-eluting stent (EES) implantation in hemodialysis (HD) patients with coronary artery disease. We enrolled 221 consecutive HD patients successfully treated with EES implantation for coronary lesions. Over the following 2 years, we assessed the incidence of target lesion revascularization (TLR) and major adverse cardiac event (MACE), defined as the composite endpoint of TLR, all-cause mortality, or myocardial infarction. We performed a propensity-score matching analysis and collected follow-up coronary angiography data. There were 91 patients in the BP-EES group and 130 in the DP-EES group. Male sex and diabetes rates were significantly lower in the BP-EES group than in the DP-EES group. A debulking device was less frequently used in the BP-EES group than in the DP-EES group (7.6% vs. 21.5%, p = 0.006). TLR occurred in 38 patients, while stent thrombosis was observed in 3 patients; 19 patients died. TLR and MACE rates at 2 years were comparable between the two groups (19.2% in the BP-EES group vs. 20.4% in the DP-EES group, p = 0.73 and 26.9% vs. 34.2%, p = 0.93, respectively). In the propensity-score-matched cohort, TLR and MACE rates were similar between the two groups (19.2% in the BP-EES group vs. 18.1% in the DP-EES group, p = 0.69, and 26.9% vs. 30.2%, p = 0.66, respectively). Restenosis rates at follow-up angiography were similar between the two groups (p = 0.79). In hemodialysis patients, BP-EES and DP-EES showed similar 2-year clinical outcomes.
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http://dx.doi.org/10.1007/s12928-021-00827-xDOI Listing
November 2021

CRISPR-Cas9 Mutagenesis in for Phenotypic Analyses in the F Generation and Beyond.

Cold Spring Harb Protoc 2021 Jul 8. Epub 2021 Jul 8.

Department of Biology, University of Virginia, Charlottesville, Virginia 22904, USA

CRISPR-Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR-Cas9 can be used to create either mosaic loss-of-function embryos that display F generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR-Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.
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http://dx.doi.org/10.1101/pdb.prot106971DOI Listing
July 2021

Outcomes after drug-coated balloon interventions for de novo coronary lesions in the patients on chronic hemodialysis.

Heart Vessels 2021 Nov 25;36(11):1646-1652. Epub 2021 Apr 25.

Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

The impact of drug-coated balloon (DCB) on hemodialysis (HD) patients with coronary lesions remains unclear. This study aimed to compare outcomes after DCB treatment between HD and non-HD patients with de novo coronary lesions. A total of 235 consecutive patients who electively underwent DCB treatment for de novo coronary lesions were included (HD group: n = 100; non-HD group: n = 135). Angiographic follow-up was performed 6 months after the procedure. Patients were clinically followed up for 2 years. The incidence rates of target lesion revascularization (TLR) and major adverse cardiac events (MACE) were investigated. Diabetes and a history of coronary bypass grafting were more frequent in the HD group than in the non-HD group (69.0% vs. 50.7%, p = 0.007, and 24.0% vs 9.1%, p = 0.013, respectively). The reference diameter and pre-procedural diameter stenosis were greater in the HD group than in the non-HD group (2.49 mm vs. 2.24 mm, p = 0.007, and 65.9% vs. 59.6%, p = 0.015, respectively). Calcification was observed in 65.5% of all lesions, and rotational atherectomy was performed in 30.2% patients. The average diameter of the DCB was 2.51 mm (2.57 mm, HD group vs. 2.47 mm, non-HD group, p = 0.14). Although post-procedural diameter stenosis was similar between the groups, late lumen loss on follow-up angiography was larger in HD patients than in non-HD patients (0.27 mm vs. - 0.03 mm, p = 0.0009). The 2-year rates of freedom from TLR and MACE were lower in HD patients than in non-HD patients [79.3% vs. 91.7%, hazard ratio (HR) 2.76, 95% confidence interval (CI) 1.23-6.77, p = 0.014; and 61.6% vs. 89.4%, HR 4.60, 95% CI 2.30-10.2, p < 0.001, respectively]. In conclusion, the rates of TLR and MACE after DCB treatment were higher in HD patients than in non-HD patients.
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http://dx.doi.org/10.1007/s00380-021-01858-3DOI Listing
November 2021

Long-term clinical outcomes after self-expandable bare nitinol stent implantation for femoropopliteal occlusive disease in hemodialysis patients.

Catheter Cardiovasc Interv 2021 02 12;97(2):318-324. Epub 2020 Nov 12.

Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Objectives: To compare the long-term clinical outcomes after self-expandable bare nitinol stent (BNS) implantation between hemodialysis (HD) and non-HD patients with femoropopliteal (FP) disease.

Background: Although a BNS has been commonly used in patients with FP disease, the long-term efficacy of BNSs in HD patients remains unknown.

Methods: In total, 427 HD patients treated with a BNS for FP disease were enrolled, along with 157 non-HD patients as a control group. Over the following 5 years, the incidence of target lesion revascularization (TLR), major amputation and mortality was investigated. We also performed propensity-score matching analysis.

Results: The 5-year TLR rate (45.2 vs. 32.5%, p = .013) and mortality rate (39.3 vs. 14.0%, p = .0002) were significantly higher in the HD group than in the non-HD group. The major amputation rate was comparable between the groups (7.2% in the HD group vs. 2.8% in the non-HD group, p = .16). In the propensity-score-matched cohort, the TLR rate, and mortality rate were remained higher in the HD group than in the non-HD group (48.9 vs. 34.1%, hazard ratio [HR] 2.11, 95% confidence interval [CI] 1.30-3.49, p = .0024, and 47.9 vs. 12.0%, HR 3.38, 95% CI 1.86-6.56, p < .0001, respectively). The adjusted amputation rate was consistently similar between the groups (1.7% in the HD group vs. 2.7% in the non-HD group, HR 0.90, 95% CI 0.26-2.99, p = .86).

Conclusions: The TLR rate and mortality at 5 years post BNS implantation for FP disease were significantly higher in HD patients than in non-HD patients, though the limb salvage rate was similar.
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http://dx.doi.org/10.1002/ccd.29372DOI Listing
February 2021

Simple embryo injection of long single-stranded donor templates with the CRISPR/Cas9 system leads to homology-directed repair in Xenopus tropicalis and Xenopus laevis.

Genesis 2020 06 11;58(6):e23366. Epub 2020 Apr 11.

School of Natural Sciences, University of Central Missouri, Warrensburg, Missouri.

We report model experiments in which simple microinjection of fertilized eggs has been used to effectively perform homology-directed repair (HDR)-mediated gene editing in the two Xenopus species used most frequently for research: X. tropicalis and X. laevis. We have used long single-stranded DNAs having phosphorothioate modifications as donor templates for HDR at targeted genomic sites using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. First, X. tropicalis tyr mutant (i.e., albino) embryos were successfully rescued: partially pigmented tadpoles were seen in up to 35% of injected embryos, demonstrating the potential for efficient insertion of targeted point mutations. Second, in order to demonstrate the ability to tag genes with fluorescent proteins (FPs), we targeted the melanocyte-specific gene slc45a2.L of X. laevis to label it with the Superfolder green FP (sfGFP), seeing mosaic expression of sfGFP in melanophores in up to 20% of injected tadpoles. Tadpoles generated by these two approaches were raised to sexual maturity, and shown to successfully transmit HDR constructs through the germline with precise targeting and seamless recombination. F1 embryos showed rescue of the tyr mutation (X. tropicalis) and tagging in the appropriate pigment cell-specific manner of slc45a2.L with sfGFP (X. laevis).
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http://dx.doi.org/10.1002/dvg.23366DOI Listing
June 2020

Aortic valve replacement via a right parasternal approach in a patient with a history of coronary artery bypass surgery and pericardiectomy: a case report.

Surg Case Rep 2019 Mar 4;5(1):39. Epub 2019 Mar 4.

Department of Cardiovascular Surgery, Nagoya Kyoritsu Hospital, 1-172 Hokke, Nakagawa, Nagoya, 454-0933, Japan.

Background: The number of patients who require aortic valve replacement after coronary artery bypass grafting continues to increase. Re-operative cardiovascular surgery after coronary artery bypass grafting has various risk factors related to median re-sternotomy. It is particularly essential to avoid damage to the living graft. We successfully performed aortic valve replacement via right parasternal thoracotomy in a patient who had undergone coronary artery bypass grafting.

Case Presentation: An 80-year-old man who had undergone coronary artery bypass grafting was referred to our hospital for syncope caused by severe aortic valve stenosis. He also had a history of pericardiotomy for constrictive pericarditis. His left internal thoracic artery bypass graft was patent. Aortic valve replacement was performed through a small right parasternal thoracotomy during cardiac arrest following cardiopulmonary bypass under moderate hypothermia and hyperkalemia by intermittent selective antegrade cardioplegia. His postoperative course was uneventful.

Conclusion: Aortic valve replacement via right parasternal thoracotomy with moderate hypothermia and hyperkalemia was safe and effective for avoidance of re-sternotomy-related complications.
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http://dx.doi.org/10.1186/s40792-019-0598-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399367PMC
March 2019

Staged coil embolization after thoracic endovascular stent grafting for aneurysmal chronic type B aortic dissection: A case report.

SAGE Open Med Case Rep 2019 12;7:2050313X19828903. Epub 2019 Feb 12.

Department of Cardiovascular Surgery, Nagoya Kyoritsu Hospital, Nagoya, Japan.

We herein describe a 38-year-old woman with Marfan syndrome and chronic type A aortic dissection. Computed tomography showed that the sinus of Valsalva and thoracoabdominal aorta had a diameter of 62 and 55 mm, respectively. After 7 months of a Bentall operation and total arch replacement with the elephant trunk technique, we performed thoracic endovascular aortic repair for an aneurysm of the descending aorta, but we preserved the retrograde flow into the false lumen because it supplied vessels perfusing the spinal cord. Computed tomography angiography 14 months after thoracic endovascular aortic repair showed that the thoracic aortic diameter had increased to 68 mm. We then performed partial (proximal only) coil embolization of the false lumen. After 6 months, the thoracic aortic diameter had decreased to 60 mm and the spinal cord remained perfused via the distal false lumen. Staged coil embolization after thoracic endovascular aortic repair for aneurysmal chronic type B aortic dissection is feasible and can be beneficial.
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http://dx.doi.org/10.1177/2050313X19828903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378514PMC
February 2019

Prognostic Risk Analyses for Postcardiotomy Extracorporeal Membrane Oxygenation in Children: A Review of Early and Intermediate Outcomes.

Pediatr Cardiol 2019 Jan 21;40(1):89-100. Epub 2018 Aug 21.

Department of Cardiovascular Surgery, Nagoya City University, Nagoya, Japan.

We evaluated the morbidity and mortality of children requiring postcardiotomy extracorporeal membrane oxygenation (ECMO) to determine independent factors affecting early and intermediate outcomes. Between January 2002 and December 2015, 79 instances of ECMO after cardiac surgery in 73 children were retrospectively reviewed. Follow-up was completed in December 2016. Predictive risk analyses were employed concerning weaning of ECMO, hospital discharge, and mortality after discharge. Age and weight were 14.9 ± 25.6 months and 7.0 ± 5.3 kg, respectively. Median support time was 8.3 ± 4.4 days. Sixty-seven (85%) were successfully weaned off ECMO and 48 (61%) survived to hospital discharge. Multi-variate logistic regression analysis identified the first day to obtain negative fluid balance after initiation of support (adjusted odds ratio = 0.42), high serum lactate levels (0.97), and high total bilirubin (0.84) during support as significant independent factors associated with successful separation from ECMO. The first day of negative fluid balance (0.65) after successful decannulation was an independent risk factor for survival to hospital discharge. After hospital discharge, actuarial 1-year, 5-year, and 10-year survival rates were 94%, 78%, and 78%, respectively. Low weight increased the risk of death after hospital discharge by a multi-variate Cox hazard model. High serum lactate, high serum bilirubin, and unable to obtain early negative fluid balance during support impacted mortality of decannulation. Obtaining a late negative fluid balance in post-ECMO were independent risk factors for death after successful weaning. Low weight affected intermediate outcomes.
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http://dx.doi.org/10.1007/s00246-018-1964-yDOI Listing
January 2019

Optimal Structure of a Plasmonic Chip for Sensitive Bio-Detection with the Grating-Coupled Surface Plasmon-Field Enhanced Fluorescence (GC-SPF).

Materials (Basel) 2017 Sep 11;10(9). Epub 2017 Sep 11.

Inorganic Functional Materials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.

Surface plasmon field-enhanced fluorescence (SPF) has been one of the powerful tools for biosensors and bioimaging. A wavelength-scale periodic structure coated with a thin metal film is called a plasmonic chip, and it can provide SPF. SPF of Cy5-streptavidin (Cy5-SA) was measured on a biotinylated plasmonic chip with a grating of 480 nm-pitch. The optimal structure of a plasmonic sensor-chip was designed for improving detection sensitivity. The silver film thickness dependence of the SPF intensity was measured under the irradiation of the top panel of a sensor chip. Furthermore, the dependence of the SPF intensity on the distance from the metal surface was also investigated. The optimal structure for the largest fluorescence enhancement factor was 150 nm-thick silver and 10 nm-thick SiO₂ layers due to the enhanced electric field (excitation field), the surface plasmon coupled emission (SPCE), and the interference effect with reflected light. The largest enhancement factor was found to be 170-fold. Furthermore, not only the largest fluorescence intensity but also stable lower background noise were found to be essential for higher-sensitive detection.
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http://dx.doi.org/10.3390/ma10091063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615717PMC
September 2017

Coffee reduces KRAS expression in Caco-2 human colon carcinoma cells via regulation of miRNAs.

Oncol Lett 2017 Jul 22;14(1):1109-1114. Epub 2017 May 22.

Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan.

Previous epidemiological studies have demonstrated that moderate coffee consumption is associated with a lower risk of certain types of cancer, particularly colon cancer. To elucidate the molecular basis for this protective action, the effect of coffee on Caco-2 human colon carcinoma cells was investigated. Low concentrations of coffee (<5%) inhibited proliferation of Caco-2 cells without affecting cell viability. Coffee also reduced KRAS proto-oncogene, GTPase (KRAS) gene expression in a dose-dependent manner; however, caffeine, caffeic acid and chlorogenic acid, three major constituents of coffee, did not exhibit this effect. Increasing the duration of coffee bean roasting increased the reduction in KRAS expression, suggesting that the active constituents responsible for this effect emerged during the roasting process. MicroRNA (miR) analysis revealed that coffee induced the expression of miR-30c and miR-96, both of which target the KRAS gene. The results of the present study suggested that daily coffee consumption may reduce KRAS activity, thereby preventing the malignant growth of colon carcinoma cells.
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http://dx.doi.org/10.3892/ol.2017.6227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494607PMC
July 2017

A peptide hormone required for Casparian strip diffusion barrier formation in Arabidopsis roots.

Science 2017 01;355(6322):284-286

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan.

Plants achieve mineral ion homeostasis by means of a hydrophobic barrier on endodermal cells called the Casparian strip, which restricts lateral diffusion of ions between the root vascular bundles and the soil. We identified a family of sulfated peptides required for contiguous Casparian strip formation in Arabidopsis roots. These peptide hormones, which we named Casparian strip integrity factor 1 (CIF1) and CIF2, are expressed in the root stele and specifically bind the endodermis-expressed leucine-rich repeat receptor kinase GASSHO1 (GSO1)/SCHENGEN3 and its homolog, GSO2. A mutant devoid of CIF peptides is defective in ion homeostasis in the xylem. CIF genes are environmentally responsive. Casparian strip regulation is not merely a passive process driven by root developmental cues; it also serves as an active strategy to cope with adverse soil conditions.
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http://dx.doi.org/10.1126/science.aai9057DOI Listing
January 2017

High variability of expression profiles of homeologous genes for Wnt, Hh, Notch, and Hippo signaling pathways in Xenopus laevis.

Dev Biol 2017 06 12;426(2):270-290. Epub 2017 Jan 12.

School of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan. Electronic address:

Cell signaling pathways, such as Wnt, Hedgehog (Hh), Notch, and Hippo, are essential for embryogenesis, organogenesis, and tissue homeostasis. In this study, we analyzed 415 genes involved in these pathways in the allotetraploid frog, Xenopus laevis. Most genes are retained in two subgenomes called L and S (193 homeologous gene pairs and 29 singletons). This conservation rate of homeologs is much higher than that of all genes in the X. laevis genome (86.9% vs 60.2%). Among singletons, 24 genes are retained in the L subgenome, a rate similar to the average for all genes (82.8% vs 74.6%). In addition, as general components of signal transduction, we also analyzed 32 heparan sulfate proteoglycan (HSPG)-related genes and eight TLE/Groucho transcriptional corepressors-related genes. In these gene sets, all homeologous pairs have been retained. Transcriptome analysis using RNA-seq data from developmental stages and adult tissues demonstrated that most homeologous pairs of signaling components have variable expression patterns, in contrast to the conservative expression profiles of homeologs for transcription factors. Our results indicate that homeologous gene pairs for cell signaling regulation have tended to become subfunctionalized after allotetraploidization. Diversification of signaling pathways by subfunctionalization of homeologs may enhance environmental adaptability. These results provide insights into the evolution of signaling pathways after polyploidization.
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http://dx.doi.org/10.1016/j.ydbio.2016.12.006DOI Listing
June 2017

Genome evolution in the allotetraploid frog Xenopus laevis.

Nature 2016 10;538(7625):336-343

US Department of Energy Joint Genome Institute, Walnut Creek, California 94598, USA.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.
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http://dx.doi.org/10.1038/nature19840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313049PMC
October 2016

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

Dev Biol 2017 06 3;426(2):472-486. Epub 2016 Sep 3.

Department of Biology, University of Virginia, Charlottesville, VA 22904, USA. Electronic address:

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.
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http://dx.doi.org/10.1016/j.ydbio.2016.08.020DOI Listing
June 2017

[Hemorrhage from the subclavian artery after lung cancer resection treated by stent graft placement].

Kyobu Geka 2015 Mar;68(3):197-200

Department of Surgery, Nagoya City East Medical Center, Nagoya, Japan.

We present a case of a 79-year-old man with right apical invasive lung cancer which was treated by induction radiotherapy followed by right upper lobectomy with chest wall resection. Four days after the operation, hemorrhage from the funicular structure in the cupula of the parietal pleura was observed, and hemostasis was achieved by ligation and fibrin sheet pasting. At the time, we were not able to detect the hemorrhage from the subclavian artery. Two days after the 1st hemostasis, hemorrhage reccurred. Hemorrhage from the inferior border of the subclavian artery was observed, and hemostasis was achieved by direct suture and fibrin sheet pasting. One day after the 2nd hemostasis, re-recurrent hemorrhage occurred. Stent graft placement was performed under local anesthesia. No hemorrhage occurred after the stent graft placement.
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March 2015

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

Dev Biol 2015 Dec 25;408(2):328-44. Epub 2015 Feb 25.

Department of Biology, University of Virginia, Charlottesville, VA 22904, USA; Department of Ophthalmology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. Electronic address:

Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans.
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http://dx.doi.org/10.1016/j.ydbio.2015.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549229PMC
December 2015

Pre-operative endostent placement to allow the complete and safe resection of a recurrent tumor that had tightly adhered to the subclavian artery: A case report.

SAGE Open Med Case Rep 2015 29;3:2050313X15583209. Epub 2015 Apr 29.

Department of Cardiovascular Surgery, Nagoya City East Medical Center, Nagoya, Japan.

Radiation and tumor infiltration confer a high risk of bleeding on surgical removal of tumor. We report on the case of a 42-year-old woman with a recurrent occult subclavian tumor in her right breast. Computed tomography revealed enhanced tumor adhesion to the subclavian artery at the infraclavicular lymph node. No other metastases were detected. We pre-operatively performed stenting of the right subclavian artery, and the tumor was resected completely and safely.
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http://dx.doi.org/10.1177/2050313X15583209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857311PMC
August 2016

Cas9-based genome editing in Xenopus tropicalis.

Methods Enzymol 2014 ;546:355-75

Department of Biology, University of Virginia, Charlottesville, Virginia, USA. Electronic address:

Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.
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http://dx.doi.org/10.1016/B978-0-12-801185-0.00017-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284096PMC
July 2015

Xenopus mutant reveals necessity of rax for specifying the eye field which otherwise forms tissue with telencephalic and diencephalic character.

Dev Biol 2014 Nov 16;395(2):317-330. Epub 2014 Sep 16.

Department of Biology, University of Virginia, Charlottesville, VA 22904, USA.

The retinal anterior homeobox (rax) gene encodes a transcription factor necessary for vertebrate eye development. rax transcription is initiated at the end of gastrulation in Xenopus, and is a key part of the regulatory network specifying anterior neural plate and retina. We describe here a Xenopus tropicalis rax mutant, the first mutant analyzed in detail from a reverse genetic screen. As in other vertebrates, this nonsense mutation results in eyeless animals, and is lethal peri-metamorphosis. Tissue normally fated to form retina in these mutants instead forms tissue with characteristics of diencephalon and telencephalon. This implies that a key role of rax, in addition to defining the eye field, is in preventing alternative forebrain identities. Our data highlight that brain and retina regions are not determined by the mid-gastrula stage but are by the neural plate stage. An RNA-Seq analysis and in situ hybridization assays for early gene expression in the mutant revealed that several key eye field transcription factors (e.g. pax6, lhx2 and six6) are not dependent on rax activity through neurulation. However, these analyses identified other genes either up- or down-regulated in mutant presumptive retinal tissue. Two neural patterning genes of particular interest that appear up-regulated in the rax mutant RNA-seq analysis are hesx1 and fezf2. These genes were not previously known to be regulated by rax. The normal function of rax is to partially repress their expression by an indirect mechanism in the presumptive retina region in wildtype embryos, thus accounting for the apparent up-regulation in the rax mutant. Knock-down experiments using antisense morpholino oligonucleotides directed against hesx1 and fezf2 show that failure to repress these two genes contributes to transformation of presumptive retinal tissue into non-retinal forebrain identities in the rax mutant.
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http://dx.doi.org/10.1016/j.ydbio.2014.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267880PMC
November 2014

[Quadricuspid aortic valve; report of two cases].

Kyobu Geka 2014 Aug;67(9):820-3

Department of Cardiovascular Surgery, Nagoya City East Medical Center, Nagoya, Japan.

Quardicupid aortic valve( QAV) is a comparatively rare congenital anomaly, which presents with frequent aortic regurgitaion ( AR) due to sclerotic changes. We report 2 cases (a 67-year-old woman and a 53-year-old man) of QAV associated with AR. We made an definite diagnosis by preoperative transthoracic and intraoperative transesophageal echocardiography. Intraoperative findings showed type C QAV in case 1 and type B QAV in case 2 according to the Hurwitz classification. The left coronary ostia was slightly shifted to the aortic root in case 1. There were no other congenital anomalies, therefore only aortic valve replacement with mechanical prosthesis was performed in both cases. The postoperative courses were uneventful.
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August 2014

[Penetrating injury of the left ventricle caused by a nail gun; report of a case].

Kyobu Geka 2014 May;67(5):411-4

Department of Cardiovascular Surgery, Nagoya East Medical Center, Nagoya, Japan.

Penetrating cardiac injuries are life-threatening emergencies. We present a case of a 24-year-old construction worker who accidentally shot himself with a nail gun. Chest X-ray showed a 6 cm-long nail overlapping the cardiac shadow. A computed tomography (CT) scan revealed the nail penetrating the left ventricle and a large amount of pericardial effusion. Median sternotomy was performed and cardiopulmomary bypass was established. Then, the nail was removed and the left ventricular wound was repaired by 4-0 Prolene mattress sutures buttressed with felt-strips. He had an uncomplicated postoperative course and was discharged 7 days postoperatively.
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May 2014

[Papillary fibroelastoma attached to the underside of the aortic valve; report of a case].

Kyobu Geka 2014 Feb;67(2):161-3

Department of Cardiovascular Surgery, Nagoya City East Medical Center, Nagoya, Japan.

We report a case of papillary fibroelastoma of the aortic valve. A 72-year-old man was referred to our hospital with cardiac tumor incidentally detected by transthoracic echocardiography. Transesophageal echocardiography showed a mobile tumor on the ventricular side of the aortic valve. We performed semi-emergency surgery and resected the tumor with a blade to prevent embolism. Pathologic findings revealed papillary fibroelastoma. The postoperative course was uneventful, and the patient was discharged 10 days arter surgery. Follow-up transthoracic echocardiography has not shown any evidence of local recurrence.
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February 2014

Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.

Genesis 2013 Dec;51(12):835-43

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.
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http://dx.doi.org/10.1002/dvg.22720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947545PMC
December 2013

Sivelestat attenuates lung injury in surgery for congenital heart disease with pulmonary hypertension.

Ann Thorac Surg 2013 Dec 25;96(6):2184-91. Epub 2013 Sep 25.

Department of Cardiovascular Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

Background: Pulmonary hypertension associated with congenital heart disease increases the risk of surgery using cardiopulmonary bypass. Sivelestat is a neutrophil elastase inhibitor thought to have a prophylactic effect against lung injury after surgery using bypass. We elucidated that Sivelestat had the protective effect on lung in patients with congenital heart disease and pulmonary hypertension who underwent surgery using bypass.

Methods: This study was a controlled prospective randomized trial and enrolled 13 neonates or infants with ventricular septal defect and pulmonary hypertension. The patients were assigned to either sivelestat with the dose of 0.2 mg/kg per hour (sivelestat group, n = 7) or saline (placebo group, n = 6) from the start of bypass until 6 hours after bypass. Proinflammatory cytokines and adhesion molecules on leukocytes were measured at 10 time points during the above period. Pulmonary function was assessed perioperatively.

Results: Compared with the placebo group, the sivelestat group had significantly lower values of alveolar-arterial oxygen tension gradient at 24 hours (p = 0.038) and at 48 hours (p = 0.028) after bypass, and significantly better balance of hydration at 48 hours after bypass (p = 0.012). The sivelestat group also showed significantly lower plasma levels of interleukin-8 immediately after bypass (p = 0.041) and interleukin-10 at 15 minutes after removal of the aortic cross-clamp (p = 0.048), and immediately after bypass (p = 0.037).

Conclusions: Administration of sivelestat during bypass prevented pulmonary damage and activities of proinflammatory cytokines at the cardiac operation in neonates or infants. Our results show that sivelestat may be considered to protect pulmonary function against the injury by bypass.
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http://dx.doi.org/10.1016/j.athoracsur.2013.07.017DOI Listing
December 2013

Initiation of carbon nanotube growth by well-defined carbon nanorings.

Nat Chem 2013 Jul 26;5(7):572-6. Epub 2013 May 26.

Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan.

Carbon nanotubes (CNTs), tubular molecular entities that consist of sp(2)-hybridized carbon atoms, are currently produced as mixtures that contain tubes of various diameters and different sidewall structures. The electronic and optical properties of CNTs are determined by their diameters and sidewall structures and so a controlled synthesis of uniform-diameter, single-chirality CNTs-a significant chemical challenge-would provide access to pure samples with predictable properties. Here we report a rational bottom-up approach to synthesize structurally uniform CNTs using carbon nanorings (cycloparaphenylenes) as templates and ethanol as the carbon source. The average diameter of the CNTs formed is close to that of the carbon nanorings used, which supports the operation of a 'growth-from-template' mechanism in CNT formation. This bottom-up organic chemistry approach is intrinsically different from other conventional approaches to making CNTs and, if it can be optimized sufficiently, offers a route to the programmable synthesis of structurally uniform CNTs.
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http://dx.doi.org/10.1038/nchem.1655DOI Listing
July 2013

Organic small hairpin RNAs (OshR): a do-it-yourself platform for transgene-based gene silencing.

Methods 2013 Sep 23;63(2):101-9. Epub 2013 May 23.

Department of Biology, Brandeis University, United States; Rosenstiel Basic Medical Science Research Center, United States.

The RNA interference (RNAi) pathway in animal cells can be harnessed to silence gene expression with artificial small interfering RNAs (siRNAs) or transgenes that express small hairpin RNAs (shRNAs). The transgene-expressing shRNA approach has been adapted into large-scale resources for genome-wide loss-of-function screens, whereas focused studies on a narrow set of genes can be achieved by using individual shRNA constructs from these resources. Although current shRNA repositories generally work, they might fail in certain situations and therefore necessitate other alternatives. We detail here a new highly-accessible and rational design of custom shRNAs that utilizes a refined backbone configuration termed the 'organic' shRNA (OshR) platform. The OshR platform is 'organic' because it conforms more naturally to the endogenous vertebrate miRNAs by maintaining specific bulges and incorporating strategic mismatches to insure the desired guide strand is produced while reducing the accumulation of passenger strands that might contribute to off-target effects. We also demonstrate that the reliability of the OshR platform for gene silencing is increased when sequences target the 3' UnTranslated Region (3'UTR) of a gene. We further compare the OshR platform with the current and emerging shRNA designs, and propose that the OshR platform is a novel approach that can allow investigators to generate custom and effective shRNAs for individual gene functional studies.
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http://dx.doi.org/10.1016/j.ymeth.2013.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966114PMC
September 2013

Roles of ADAM13-regulated Wnt activity in early Xenopus eye development.

Dev Biol 2012 Mar 28;363(1):147-54. Epub 2011 Dec 28.

Department of Cell Biology and the Morphogenesis and Regenerative Medicine Institute, University of Virginia Health System, Charlottesville, VA 22908, USA.

Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in Xenopus tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus.
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http://dx.doi.org/10.1016/j.ydbio.2011.12.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288294PMC
March 2012

Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus.

Genesis 2012 Mar 27;50(3):307-15. Epub 2011 Dec 27.

Department of Biology, University of Virginia, Charlottesville, VA 22904, USA.

We have developed a method of injecting bacterial artificial chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.
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http://dx.doi.org/10.1002/dvg.20819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870158PMC
March 2012

An aneurysm at the cannulation site discovered 40 years after cardiac surgery: report of a case.

Ann Thorac Cardiovasc Surg 2008 Aug;14(4):267-9

Department of Cardiovascular Surgery, Nagoya City University Graduate School of Medical Sciences, Mizuho-cho, Mizuho-ku, Nagoya, Japan.

We report the successful surgical treatment of a pseudoaneurysm of the ascending aorta in a 45-year-old man who underwent surgical closure of a ventricular septal defect at the age of 5. A computed tomography scan ordered for the investigation of a pulmonary mass happened to detect a pseudoaneurysm (20 mm diameter). The pseudoaneurysm protruded anteriorly from the ascending aorta at the previous aortic cannulation site. The ascending aorta, including the lesion, was replaced with a prosthetic graft uneventfully. The cause of the pseudoaneurysm was considered iatrogenic, since he had no prior history of postoperative mediastinitis or blunt chest trauma. A computed tomography scan is a useful means to detect a mass on the ascending aorta following cardiac surgery, and it provides important information to help define a surgical strategy.
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August 2008

Surgical treatment for infected thrombus in the superior vena cava using an off-pump venoatrial shunt.

Ann Thorac Surg 2008 Mar;85(3):1113-4

Department of Cardiovascular Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.

We report a septic patient who had an infected thrombus that extended from the right internal jugular vein to the right atrium 1 cm below the superior venocaval junction. The thrombus was successfully removed using an off-pump shunt placed between the innominate vein and the right atrium.
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http://dx.doi.org/10.1016/j.athoracsur.2007.08.031DOI Listing
March 2008
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