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Aoshima Keisuke

Faculty of Veterinary Medicine　Veterinary Medicine　Clinical SciencesLecturer
One Health Research CenterLecturer

Researcher basic information

■ Degree
  • DVM, Hokkaido University, Apr. 2011
  • PhD, Hokkaido University, Mar. 2016
  • DJCVP, Japanese College of Veterinary Pathologist, Apr. 2016
■ URL
researchmap URLホームページURL■ Various IDs
ORCID IDJ-Global ID■ Research Keywords and Fields
Research Field
  • Life Science, Molecular biology, Epigenetics
  • Life Science, Tumor biology, Hemangiosarcoma
  • Life Science, Veterinary medical science, Veterinary pathology
■ Educational Organization

Career

■ Career
Career
  • Oct. 2023 - Present
    Hokkaido University, Faculty of Veterinary Medicine, Senior Lecture
  • Apr. 2017 - Sep. 2023
    Hokkaido University,, Faculty of Veterinary Medicine,, Assistant Professor
  • Apr. 2018 - May 2020
    Yale School of Medicine, Department of Pathology, Visiting Research Scientist, United States
  • Nov. 2015 - Mar. 2017
    Hokkaido University,, Graduate School of Veterinary Medicine,, Assistant Professor
  • Jun. 2014 - Oct. 2015
    Hokkaido University,, Graduate School of Veterinary Medicine,, Specially Appointed Assistant Professor
  • Apr. 2013 - Jun. 2014
    Hokkaido University,, Graduate School of Veterinary Medicine, Research Fellow of Japan Society for the Promotion of Science
Educational Background
  • Apr. 2011 - Jun. 2014, Hokkaido University,, Graduate School of Veterinary Medicine
  • Feb. 2012 - Feb. 2014, The University of Tokyo,, Graduate School of Agricultural and Life Sciences,, Research Student
  • Jun. 2011 - Jan. 2012, Kyoto University,, Graduate School of Medicine,, Research Student
  • Apr. 2005 - Mar. 2011, Hokkaido University,, School of Veterinary Medicine

Research activity information

■ Awards
  • Sep. 2014, The 2nd Sapporo Summer Seminar for One Health., Special Award
    Keisuke Aoshima
  • Mar. 2011, Japan Veterinary Medical Association President's Award
    Keisuke Aoshima
  • Jun. 2006, Hokkaido University, Nitobe Prize
    Keisuke Aoshima
■ Papers
  • Lactate dehydrogenase is associated with cholesterol/lipid metabolism, and fluvastatin plus dipyridamole suppresses canine hemangiosarcoma growth in patient-derived xenograft models
    Tamami Suzuki; Suzune Tanaka; Keika Kishimoto; Takuma Goto; Jumpei Yamazaki; Takashi Kimura; Keisuke Aoshima
    bioRxiv, 05 Mar. 2026, [Last author, Corresponding author]
    51350725;36887866
  • Lysine lactylation regulates ATF4-mediated stress responses under glucose starvation in canine hemangiosarcoma
    Tamami Suzuki; Kazuki Heishima; Jumpei Yamazaki; Masaya Yamazaki; Ryohei Kinoshita; Sangho Kim; Kenji Hosoya; Yuko Okamatsu-Ogura; Michihito Sasaki; Peng Xu; Qin Yan; Takashi Kimura; Keisuke Aoshima
    Frontiers in Veterinary Science, 13, Frontiers Media SA, 12 Feb. 2026, [Peer-reviewed], [Last author, Corresponding author]
    English, Scientific journal, Excess lactate is produced in tumor cells by aerobic glycolysis and regulates gene expressions by histone lactylation. However, how histone lactylation functions under glucose-limited conditions remains unknown. Here, we show that lysine lactylation redistributes to transcription start sites (TSSs) during glucose deprivation, thereby altering biological behaviors in canine hemangiosarcoma (HSA) cells. Glucose deprivation significantly decreased global histone lactylation levels, while lactylation peaks were enriched at TSSs of ATF4-regulated stress-response, asparagine-synthesis and immune-related genes. Stress-response gene expressions were upregulated, and ATF4 polyclonal knockout abrogated this activation. [U- 13 C]glutamine tracing demonstrated that HSA cells synthesized asparagine from glutamine when glucose was scarce, and asparagine supplementation modestly activated cell proliferation. In HSA patient tissues, H3K18la levels were heterogeneous, and M2-like macrophages preferentially infiltrated tumor regions showing low histone lactylation levels. These findings demonstrate that lysine lactylation regulates transcription that supports tumor cell survival and fosters a pro-tumor microenvironment even under glucose-limited conditions., 36887866
  • Calcinosis circumscripta of the extremities and the tongue with multifocal arterial calcification secondary to chronic kidney disease in a cat.
    Ai Koshikawa; Shingo Miki; Sangho Kim; Takashi Kimura; Keisuke Aoshima
    The Journal of veterinary medical science, 18 Jul. 2025, [Peer-reviewed], [Last author, Corresponding author], [Domestic magazines]
    English, Scientific journal, A 3-year-old neutered male mixed-breed cat was presented with right urinary tract obstruction caused by a urolith and severe atrophy of the left kidney. In the next year and a half, chronic kidney disease (CKD) progressed, and several masses were identified in the extremities, along with an ulcer on the tongue. Histologically, the extremity masses consisted of calcium deposits separated by fibrovascular septa, and the tongue ulcer showed severe neutrophilic infiltration with pervasive calcium deposition. These lesions were diagnosed as calcinosis circumscripta (CCs). Concurrently, multifocal mineralization of the aorta and of small- to medium-sized arteries in several organs indicated widespread vascular calcification. Both extremity and vascular lesions were likely secondary to the excessive calcium-phosphate product caused by CKD. Although CCs in either the extremities or tongue has been reported in cats, this case is notable for the presence of CCs in both extremities and tongue with systemic vascular calcification.
  • Progressive myelomalacia with spinal cord disorders along with severe intradural hemorrhage in a cat.
    Takafumi Sunaga; Shingo Miki; Fuka Takahashi; Keisuke Aoshima; Sangho Kim; Masahiro Okumura
    The Journal of veterinary medical science, 02 Jul. 2025, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, Progressive myelomalacia (PMM) is a severe neurological disorder. Although several case reports have been published, PMM is uncommon in cats. A 9-year-old neutered male domestic short-haired cat presented with hindlimb paraplegia. Based on the neurological examination, severe abnormalities in the L4-S3 segment were suspected. Based on magnetic resonance imaging findings, intervertebral disk herniation (IVDH) was suspected at the L5-L6 intervertebral disk. A hemilaminectomy and durotomy were performed. Four days after surgery, PMM was suspected, and the cat was euthanized. An autopsy was performed, and a histopathological examination confirmed PMM secondary to severe spinal disorders.
  • Ultrasonographic features and blood biochemistry findings of cholangiocarcinoma in a captive polar bear (Ursus maritimus)
    Keita Kakisaka; Nozomu Yokoyama; Fuka Takahashi; Keisuke Aoshima; Mitsuyoshi Takiguchi
    Veterinary Record Case Reports, Wiley, 28 Jun. 2025, [Peer-reviewed]
    English, Scientific journal, ABSTRACT

    A 29‐year‐old male captive polar bear (Ursus maritimus) with a year‐long history of elevated gamma‐glutamyl transpeptidase (GGT) presented with a 2‐week history of anorexia and abdominal distention. Blood biochemistry revealed a severely elevated GGT (>1500 IU/L). Abdominal ultrasound revealed multiple cystic hepatic masses and marked ascites. Fourteen days after initial presentation, the patient was found dead. Postmortem examination revealed hepatomegaly with diffuse, multifocal, coalescing cysts containing a red‒tan to dark‐red serous fluid. Histopathologically, the cyst walls were lined by flattened or cuboidal neoplastic cells, some exhibiting tubulopapillary projections into the lumen. Metastatic lesions were identified in the anterior mediastinal lymph nodes, lungs and kidneys. This case was diagnosed as cholangiocarcinoma. While there are numerous reports of cholangiocarcinoma in polar bears, published data on blood biochemistry and abdominal ultrasonographic findings are limited. This case report illustrates the benefit of blood biochemistry and abdominal ultrasound in investigating cholangiocarcinoma in polar bears.
  • Nodal T-cell lymphoma with eosinophilic infiltration and sclerosing fibroplasia in a cat with eosinophilia.
    Tamami Suzuki; Jumpei Yamazaki; Kouta Yamaguchi; Keisuke Aoshima; Takashi Kimura
    The Journal of veterinary medical science, 86, 12, 1252, 1255, 01 Dec. 2024, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, An 8-year-old castrated male mixed-breed cat presented with an abdominal mass of unknown origin, accompanied by eosinophilia. Autopsy revealed mild-to-severe enlargement of lymph nodes throughout the body and multiple nodules in the lungs. Histopathologically, the lymph nodes showed severe fibroplasia and infiltration by a large number of eosinophils and fewer tumor cells, exhibiting large-sized lymphoid cell morphology. Metastatic lesions of tumor cells with eosinophilic infiltration and fibrosis were observed in the lungs, liver, kidneys, stomach, and intestines. Immunohistochemistry revealed that the tumor cells were positive for CD3 and negative for B cell and mast cell markers. Thus, T-cell lymphoma with eosinophilic infiltration and sclerosing fibroplasia was diagnosed.
  • First molecular characterization of Burkholderia mallei strains isolated from horses in Mongolia.
    Yoshiki Ichikawa; Liushiqi Borjigin; Batchuluun Enkhtuul; Ochirbat Khurtsbaatar; Keisuke Aoshima; Atsushi Kobayashi; Vanaabaatar Batbaatar; Takashi Kimura
    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 123, 105616, 105616, Elsevier BV, Sep. 2024, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Glanders, a highly contagious and often fatal disease affecting equids, is caused by Burkholderia mallei. Although sporadic cases of equine glanders have recently been documented in Mongolia, genome sequencing and molecular studies of the bacteria within this region are lacking. This study provided the first molecular characterization of B. mallei isolated from four native Mongolian horses from two different provinces in 2019 and 2022 by applying whole-genome sequencing with two SNP types (previously developed genotyping with 15 SNP markers that provide global coverage of the B. mallei population and the core genome coding SNP typing developed in this study). The Mongolian isolates were located within the L3B1 cluster, which was previously associated with the V-120 strain from Russia. Within the L3B1 cluster shared by neighboring countries, they were in a unique subbranch. In this study, specific SNP markers unique to the Mongolian strains were identified to track these strains using a high-resolution melting analysis (HRMA). This study revealed the unique phylogenetic background of Mongolian strains isolated from the eastern part of Mongolia. HRMA specific to the Mongolian subbranch may contribute to the molecular epidemiological monitoring of glanders in Mongolia and surrounding countries.
  • FOUR-WEEK ORAL ADMINISTRATION OF BALOXAVIR MARBOXIL AS AN ANTI-INFLUENZA VIRUS DRUG SHOWS NO TOXICITY IN CHICKENS.
    Mariko Miki; Ryo Daniel Obara; Kyohei Nishimura; Takao Shishido; Yoshinori Ikenaka; Ryoko Oka; Kenji Sato; Shouta M M Nakayama; Takashi Kimura; Atsushi Kobayashi; Keisuke Aoshima; Keisuke Saito; Takahiro Hiono; Norikazu Isoda; Yoshihiro Sakoda
    Journal of zoo and wildlife medicine : official publication of the American Association of Zoo Veterinarians, 55, 2, 313, 321, Jun. 2024, [Peer-reviewed], [International Magazine]
    English, Scientific journal, High pathogenicity avian influenza is an acute zoonotic disease with high mortality in birds caused by a high pathogenicity avian influenza virus (HPAIV). Recently, HPAIV has rapidly spread worldwide and has killed many wild birds, including endangered species. Baloxavir marboxil (BXM), an anti-influenza agent used for humans, was reported to reduce mortality and virus secretion from HPAIV-infected chickens (Gallus domesticus, order Galliformes) at a dosage of ≥2.5 mg/kg when administered simultaneously with viral challenge. Application of this treatment to endangered birds requires further information on potential avian-specific toxicity caused by repeated exposure to BXM over the long term. To obtain information of potential avian-specific toxicity, a 4-wk oral repeated-dose study of BXM was conducted in chickens (n = 6 or 7 per group), which are commonly used as laboratory avian species. The study was conducted in reference to the human pharmaceutical guidelines for nonclinical repeated-dose drug toxicity studies to evaluate systemic toxicity and exposure. No adverse changes were observed in any organs examined, and dose proportional increases in systemic exposure to active pharmaceutical ingredients were noted from 12.5 to 62.5 mg/kg per day. BXM showed no toxicity to chickens at doses of up to 62.5 mg/kg per day, at which systemic exposure was approximately 71 times higher than systemic exposure at 2.5 mg/kg, the reported efficacious dosage amount, in HPAIV-infected chickens. These results also suggest that BXM could be considered safe for treating HPAIV-infected endangered birds due to its high safety margin compared with the efficacy dose. The data in this study could contribute to the preservation of endangered birds by using BXM as a means of protecting biodiversity.
  • Current understanding of comparative pathology and prospective research approaches for canine hemangiosarcoma.
    Tamami Suzuki; Michael James Henshaw; Teruki Yanagi; Keisuke Aoshima
    Research in veterinary science, 167, 105120, 105120, Feb. 2024, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
    English, Scientific journal, Hemangiosarcoma (HSA) is a malignant tumor originating from endothelial cells. HSA typically develops in dogs, but is rare in other animals, including humans. Although surgery and chemotherapy are conventional treatments for HSA, neither treatment can significantly improve patient prognosis. To develop novel and effective therapeutics, a deeper understanding of HSA pathogenesis must be acquired. However, the limited research tools for HSA have been unable to make a breakthrough; therefore, it is crucial to widely utilize or establish novel research tools such as patient-derived xenograft models, organoids, and chicken embryo xenograft models. The pathogenesis of the human counterpart of HSA, angiosarcoma (AS), also remains incompletely understood, preventing the extrapolation of findings from humans to dogs, unlike other diseases. In this review, we summarize the clinicopathological and morphological features of HSA, and then we discuss the current understanding of the molecular pathology of HSA. Finally, we highlight promising research tools that may accelerate HSA basic research toward developing novel therapeutics. We also briefly summarize AS to help researchers comprehend HSA from the perspective of comparative pathology.
  • Adrenocortical hypoperfusion detected by contrast-enhanced ultrasound in a dog with trilostane-induced hypoadrenocorticism.
    N Nagata; K Aoshima; K Nakamura; M Takiguchi
    The Journal of small animal practice, 64, 11, 722, 726, Nov. 2023, [Peer-reviewed], [International Magazine]
    English, A 12-year-old neutered male Chihuahua dog was diagnosed with pituitary-dependent hypercortisolism and treated with trilostane. Eighty-nine days later, the dog showed lethargy accompanied by hyponatraemia and hyperkalaemia. Hypoadrenocorticism due to trilostane was suspected, but the result of the adrenocorticotropic hormone stimulation test was not conclusive. Contrast-enhanced ultrasound showed loss of adrenocortical blood flow in both adrenal glands, indicating adrenocortical hypoperfusion and isolated hypoadrenocorticism. Treatment with fludrocortisone acetate improved the condition and electrolyte abnormalities. Thirteen months later, the dog showed alopecia, and an adrenocorticotropic hormone stimulation test revealed increased cortisol concentration, indicating hypercortisolism recurrence. The dog died due to progressive deterioration 22 months after the initial presentation. Post-mortem examination revealed focally extensive necrosis with marked calcification in the parenchyma of the adrenal glands and regeneration of the cells in the zona fasciculata with severe fibrosis. Adrenocortical hypoperfusion detected by contrast-enhanced ultrasound can support the diagnosis of adrenal necrosis and hypoadrenocorticism.
  • Diverse genome-wide DNA methylation alterations in canine hepatocellular tumours.
    Yu Asari; Jumpei Yamazaki; Oo Thandar; Tamami Suzuki; Keisuke Aoshima; Kyosuke Takeuchi; Ryohei Kinoshita; Sangho Kim; Kenji Hosoya; Teita Ishizaki; Yumiko Kagawa; Jaroslav Jelinek; Shoko Yokoyama; Noboru Sasaki; Hiroshi Ohta; Kensuke Nakamura; Mitsuyoshi Takiguchi
    Veterinary medicine and science, 22 Jul. 2023, [Peer-reviewed], [International Magazine]
    English, Scientific journal, BACKGROUND: Canine hepatocellular tumours (HCTs) are common primary liver tumours. However, the exact mechanisms of tumourigenesis remain unclear. Although some genetic mutations have been reported, DNA methylation alterations in canine HCT have not been well studied. OBJECTIVES: In this study, we aimed to analyse the DNA methylation status of canine HCT. METHODS: Tissues from 33 hepatocellular carcinomas, 3 hepatocellular adenomas, 1 nodular hyperplasia, 21 non-tumour livers from the patients and normal livers from 5 healthy dogs were used. We analysed the DNA methylation levels of 72,367 cytosine-guanine dinucleotides (CpG sites) in all 63 samples. RESULTS AND CONCLUSIONS: Although a large fraction of CpG sites that were highly methylated in the normal liver became hypomethylated in tumours from most patients, we also found some patients with less remarkable change or no change in DNA methylation. Hierarchical clustering analysis revealed that 32 of 37 tumour samples differed from normal livers, although the remaining 5 tumour livers fell into the same cluster as normal livers. In addition, the number of hypermethylated genes in tumour livers varied among tumour cases, suggesting various DNA methylation patterns in different tumour groups. However, patient and clinical parameters, such as age, were not associated with DNA methylation status. In conclusion, we found that HCTs undergo aberrant and diverse patterns of genome-wide DNA methylation compared with normal liver tissue, suggesting a complex epigenetic mechanism in canine HCT.
  • A point mutation in GPI-attachment signal peptide accelerates the development of prion disease.
    Atsushi Kobayashi; Tetsuya Hirata; Taishi Shimazaki; Yoshiko Munesue; Keisuke Aoshima; Takashi Kimura; Junko Nio-Kobayashi; Rie Hasebe; Atsuko Takeuchi; Yuichi Matsuura; Satoshi Kusumi; Daisuke Koga; Yasushi Iwasaki; Taroh Kinoshita; Shirou Mohri; Tetsuyuki Kitamoto
    Acta neuropathologica, 145, 5, 637, 650, 06 Mar. 2023, [International Magazine]
    English, Scientific journal, A missense variant from methionine to arginine at codon 232 (M232R) of the prion protein gene accounts for ~ 15% of Japanese patients with genetic prion diseases. However, pathogenic roles of the M232R substitution for the induction of prion disease have remained elusive because family history is usually absent in patients with M232R. In addition, the clinicopathologic phenotypes of patients with M232R are indistinguishable from those of sporadic Creutzfeldt-Jakob disease patients. Furthermore, the M232R substitution is located in the glycosylphosphatidylinositol (GPI)-attachment signal peptide that is cleaved off during the maturation of prion proteins. Therefore, there has been an argument that the M232R substitution might be an uncommon polymorphism rather than a pathogenic mutation. To unveil the role of the M232R substitution in the GPI-attachment signal peptide of prion protein in the pathogenesis of prion disease, here we generated a mouse model expressing human prion proteins with M232R and investigated the susceptibility to prion disease. The M232R substitution accelerates the development of prion disease in a prion strain-dependent manner, without affecting prion strain-specific histopathologic and biochemical features. The M232R substitution did not alter the attachment of GPI nor GPI-attachment site. Instead, the substitution altered endoplasmic reticulum translocation pathway of prion proteins by reducing the hydrophobicity of the GPI-attachment signal peptide, resulting in the reduction of N-linked glycosylation and GPI glycosylation of prion proteins. To the best of our knowledge, this is the first time to show a direct relationship between a point mutation in the GPI-attachment signal peptide and the development of disease.
  • Virological, pathological, and glycovirological investigations of an Ezo red fox and a tanuki naturally infected with H5N1 highly pathogenic avian influenza viruses in Hokkaido, Japan
    Takahiro Hiono; Daiki Kobayashi; Atsushi Kobayashi; Tamami Suzuki; Yuki Satake; Rio Harada; Keita Matsuno; Mariko Sashika; Hinako Ban; Maya Kobayashi; Keisuke Aoshima; Fumihito Takaya; Hiroko Fujita; Norikazu Isoda; Takashi Kimura; Yoshihiro Sakoda
    Virology, 578, 35, 44, Jan. 2023, [Peer-reviewed], [International Magazine]
    English, Scientific journal, In winter/spring 2021-2022, high pathogenicity avian influenza viruses (HPAIVs) that are genetically closely related to each other were detected worldwide. In a public garden in Sapporo, Hokkaido, Japan, a crow die-off by HPAIV infection occurred from March 29 to May 18, 2022. During the event, H5N1 HPAIVs were isolated from an Ezo red fox (Vulpes vulpes schrencki) and a tanuki (Nyctereutes procyonoides albus) found in the same garden. The fox showed viral meningoencephalitis and moderate virus replication in the upper respiratory tract, whereas the tanuki showed viral conjunctivitis and secondary bacterial infection in the eyes accompanied with visceral larva migrans. Viruses isolated from the fox and the tanuki were genetically closely related to those isolated from crows in the same garden. Various α2-3 sialosides were found in the respiratory tracts of these canid mammals, consistent with HPAIV infections in these animals. This study highlighted the importance of monitoring HPAIV infections in wild carnivore mammals to detect the potential virus spreading in nature.
  • Transcriptome and proteome analysis of dogs with precursor targeted immune-mediated anemia treated with splenectomy.
    Mei Sugawara-Suda; Keitaro Morishita; Osamu Ichii; Takashi Namba; Keisuke Aoshima; Yumiko Kagawa; Sangho Kim; Kenji Hosoya; Nozomu Yokoyama; Noboru Sasaki; Kensuke Nakamura; Jumpei Yamazaki; Mitsuyoshi Takiguchi
    PloS one, 18, 5, e0285415, 2023, [International Magazine]
    English, Scientific journal, Precursor-targeted immune-mediated anemia (PIMA) in dogs is characterized by persistent non-regenerative anemia and ineffective erythropoiesis, and it is suspected to be an immune-mediated disease. Most affected dogs respond to immunosuppressive therapies; however, some are resistant. In this study, we carried out splenectomy as an alternative therapy for refractory PIMA in dogs, and analyzed gene expression levels in the spleen of dogs with or without PIMA and in serum before and after splenectomy. A total of 1,385 genes were found to express differentially in the spleens from dogs with PIMA compared with healthy dogs by transcriptome analysis, of which 707 genes were up-regulated, including S100A12, S100A8, and S100A9 that are linked directly to the innate immune system and have been characterized as endogenous damage-associated molecular patterns. Furthermore, immunohistochemistry confirmed that S100A8/A9 protein expression levels were significantly higher in dogs with PIMA compared with those in healthy dogs. A total of 22 proteins were found to express differentially between the serum samples collected before and after splenectomy by proteome analysis, of which 12 proteins were up-regulated in the samples before. The lectin pathway of complement activation was identified by pathway analysis in pre-splenectomy samples. We speculated that S100A8/9 expression may be increased in the spleen of dogs with PIMA, resulting in activation of the lectin pathway before splenectomy. These findings further our understanding of the pathology and mechanisms of splenectomy for PIMA.
  • An experimental challenge model for Leishmania donovani in beagle dogs, showing a similar pattern of parasite burden in the peripheral blood and liver.
    Hiroya Konno; Nozomu Yokoyama; Yu Tamura; Keisuke Aoshima; Ryo Nakao; Mitsuyoshi Takiguchi; Ken Katakura
    Parasitology research, 121, 12, 3569, 3579, 12 Oct. 2022, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Leishmania donovani and Leishmania infantum are closely related species. However, the former is considered the causative agent for anthroponotic visceral leishmaniasis (AVL), while the latter is known to be responsible for zoonotic visceral leishmaniasis (ZVL) with dogs as the main reservoir host. Although molecular detection of L. donovani from naturally infected dogs has been reported in AVL endemic areas, the experimental infection of dogs with this species is very limited. Here, we constructed an experimental canine visceral leishmaniasis (CVL) model with L. donovani infection using beagle dogs. During an observation period of 8 months after parasite inoculation, few clinical symptoms were observed in the three inoculated dogs. The overall hematological and biochemical data of the dogs showed normal levels, and there were no remarkable changes in the peripheral CD4+, CD8+, CD25+, or FoxP3+ T cell populations. Liver biopsy sampling was conducted to monitor the parasite burden in the liver. A similar pattern of the amount of mitochondrial kinetoplast DNA was observed in the peripheral blood and liver by real-time PCR analysis. In addition, parasite antigens were detected from the liver biopsy sections by immunohistochemical analysis, further supporting the existence of parasites in the liver. These results showed a subclinical CVL model for L. donovani in beagle dogs with a similar kinetics of parasite burden in the peripheral blood and liver.
  • 犬尿路上皮癌におけるPDXモデルの樹立
    森下 大暉; 木之下 怜平; 青島 圭佑; 細谷 謙次; 山崎 淳平; 滝口 満喜
    日本獣医学会学術集会講演要旨集, 165回, [B2P, 03], (公社)日本獣医学会, Sep. 2022
    Japanese
  • 鼻疽の診断抗原の探索
    市川 世識; 飯沼 由希帆; 岡川 朋弘; 前川 直也; 村田 史郎; 今内 覚; 木下 優太; 丹羽 秀和; 青島 圭佑; 小林 篤史; 大橋 和彦; 木村 享史
    日本獣医学会学術集会講演要旨集, 165回, [DI1A, 09], (公社)日本獣医学会, Sep. 2022
    Japanese
  • Malignant oligoastrocytoma in the spinal cord of a cat
    Dai HASEGAWA; Keisuke AOSHIMA*; Kazuyoshi SASAOKA; Atsushi KOBAYASHI; Mitsuyoshi TAKIGUCHI; Takashi KIMURA; *Corresponding author
    Journal of Veterinary Medical Science, 84, 9, 1277, 1282, Japanese Society of Veterinary Science, Jul. 2022, [Peer-reviewed], [Corresponding author], [Domestic magazines]
    English, Scientific journal, A 12-year and 3-month spayed female mixed cat was presented with severe lumbar pain. Magnetic resonance imaging and postmortem examination revealed a swollen lesion in the spinal cord at L3 level. Histologic examination identified extensive neoplastic cell proliferation with massive necrosis in the tumor tissue. Two types of neoplastic cells were recognized. One type of neoplastic cells were large cells characterized by round to polygonal shape and abundant eosinophilic cytoplasm (referred to as "large cells"). The other neoplastic cells were small, densely proliferated, and had round to irregular shape and scant eosinophilic cytoplasm (referred to as "small cells"). Both types of cells were positive for oligodendrocyte transcription factor 2 and SRY-box transcription factor 10. Glial fibrillary acidic protein was positive in large cells but negative in most small cells. Digital analysis for Ki-67-stained tumor tissues found that total 21.1% ± 6.5% of tumor cells were positive for Ki-67. Based on these findings, we diagnosed malignant oligoastrocytoma in the spinal cord.
  • The expression of histone lysine demethylase 2B in canine hemangiosarcoma is associated with disease progression
    Kevin Christian M. Gulay; Keisuke Aoshima; Sangho Kim; Ryusei Kitaguchi; Atsushi Kobayashi; Takashi Kimura
    Veterinary and Comparative Oncology, 20, 2, 529, 534, Wiley, Jun. 2022, [Peer-reviewed], [Corresponding author]
    Scientific journal, 26148785
  • Manipulating Histone Acetylation Leads to Antitumor Effects in Hemangiosarcoma Cells.
    Tamami Suzuki; Keisuke Aoshima*; Jumpei Yamazaki; Atsushi Kobayashi; Takashi Kimura; *Corresponding author
    Veterinary and Comparative Oncology, 14 May 2022, [Peer-reviewed], [Corresponding author], [International Magazine]
    English, Scientific journal, Canine hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. HSA cell lines treated with SAHA and VPA upregulated inflammatory-related genes and attracted macrophage cell line RAW264 cells, which suggests that SAHA and VPA can affect immune responses. JQ1 stimulated autophagy and inhibited the cell cycle in HSA cell lines. Finally, we demonstrated that JQ1 suppressed HSA tumor cell proliferation in vivo although SAHA and VPA did not affect tumor growth. These results suggest that BETi can be alternative drugs for HSA treatment. Although further research is required, our study indicated that dysregulation of histone acetylation is likely to be involved in HSA malignancy. This article is protected by copyright. All rights reserved., 26148785
  • Safety Assessment of Ultrasound-Assisted Intravesical Chemotherapy in Normal Dogs: A Pilot Study
    Noboru Sasaki; Yoshinori Ikenaka; Keisuke Aoshima; Teiichiro Aoyagi; Nobuki Kudo; Kensuke Nakamura; Mitsuyoshi Takiguchi
    Frontiers in Pharmacology, 13, 837754, 837754, Frontiers Media {SA}, 18 Mar. 2022, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Intravesical chemotherapy after transurethral resection is a treatment option in patients with non-muscle invasive bladder cancer. The efficacy of intravesical chemotherapy is determined by the cellular uptake of intravesical drugs. Therefore, drug delivery technologies in the urinary bladder are promising tools for enhancing the efficacy of intravesical chemotherapy. Ultrasound-triggered microbubble cavitation may enhance the permeability of the urothelium, and thus may have potential as a drug delivery technology in the urinary bladder. Meanwhile, the enhanced permeability may increase systemic absorption of intravesical drugs, which may increase the adverse effects of the drug. The aim of this preliminary safety study was to assess the systemic absorption of an intravesical drug that was delivered by ultrasound-triggered microbubble cavitation in the urinary bladder of normal dogs. Pirarubicin, a derivative of doxorubicin, and an ultrasound contrast agent (Sonazoid) microbubbles were administered in the urinary bladder. Ultrasound (transmitting frequency 5 MHz; pulse duration 0.44 μsec; pulse repetition frequency 7.7 kHz; peak negative pressure −1.2 MPa) was exposed to the bladder using a diagnostic ultrasound probe (PLT-704SBT). The combination of ultrasound and microbubbles did not increase the plasma concentration of intravesical pirarubicin. In addition, hematoxylin and eosin staining showed that the combination of ultrasound and microbubble did not cause observable damages to the urothelium. Tissue pirarubicin concentration in the sonicated region was higher than that of the non-sonicated region in two of three dogs. The results of this pilot study demonstrate the safety of the combination of intravesical pirarubicin and ultrasound-triggered microbubble cavitation, that is, ultrasound-assisted intravesical chemotherapy.
  • Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages
    Kevin Christian M. Gulay; Keisuke Aoshima*; Naoya Maekawa; Tamami Suzuki; Satoru Konnai; Atsushi Kobayashi; Takashi Kimura; *Corresponding author
    Scientific Reports, 12, 1, 2124, 2124, Springer Science and Business Media {LLC}, 08 Feb. 2022, [Peer-reviewed], [Corresponding author], [International Magazine]
    English, Scientific journal, AbstractHemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. Tumor-associated macrophages are one of the major components of tumor microenvironment and crucial for cancer development. The presence and function of macrophages in HSA have not been studied because there is no syngeneic model for HSA. In this study, we evaluated two mouse HSA cell lines and one immortalized mouse endothelial cells for their usefulness as syngeneic models for canine HSA. Our results show that the ISOS-1 cell line develops tumors with similar morphology to canine HSA. ISOS-1 cells highly express KDM2B and have similar KDM2B target expression patterns with canine HSA. Moreover, we determine that in both ISOS-1 and canine HSA tumors, macrophages are present as a major constituent of the tumor microenvironment. These macrophages are positive for CD204, an M2 macrophage marker, and express PD-L1. ISOS-1-conditioned medium can induce M2 polarization and PD-L1 expression in RAW264.7 mouse macrophage cell line. These results show that ISOS-1 can be used as a syngenic model for canine HSA and suggest that macrophages play an important role in immune evasion in HSA. Using the syngeneic mouse model for canine HSA, we can further study the role of immune cells in the pathology of HSA., 26148785;29924181
  • CECR2 drives breast cancer metastasis by promoting NF-κB signaling and macrophage-mediated immune suppression
    Meiling Zhang; Zongzhi Z. Liu; Keisuke Aoshima; Wesley L. Cai; Hongyin Sun; Tianrui Xu; Yangyi Zhang; Yongyan An; Jocelyn F. Chen; Lok Hei Chan; Asako Aoshima; Sabine M. Lang; Zhenwei Tang; Xuanlin Che; Yao Li; Sara J. Rutter; Veerle Bossuyt; Xiang Chen; Jon S. Morrow; Lajos Pusztai; David L. Rimm; Mingzhu Yin; Qin Yan
    Science Translational Medicine, 02 Feb. 2022, [Peer-reviewed]
    Scientific journal
  • Manipulating Histone Acetylation Leads to Adverse Effects in Hemangiosarcoma Cells
    Tamami Suzuki; Keisuke Aoshima; Jumpei Yamazaki; Atsushi Kobayashi; Takashi Kimura
    bioRxiv, 11 Dec. 2021, [Corresponding author]
    26148785
  • Systemic mucoid degeneration of the arterial tunica intima in a young dog
    Yoshiki Ichikawa; Mizuki Heishima; Kristin Vyhnal; Keisuke Aoshima*; Kazuyoshi Sasaoka; Ryohei Kinoshita; Atsushi Kobayashi; Mitsuyoshi Takiguchi; Takashi Kimura; *Corresponding author
    Journal of Veterinary Diagnostic Investigation, 34, 1, 104063872110425, 104063872110425, {SAGE} Publications, 04 Sep. 2021, [Peer-reviewed], [Corresponding author]
    Scientific journal, A 27-mo-old, spayed female mixed-breed dog was presented with left forelimb pain, which progressed to full thickness necrosis of the soft tissues of multiple limbs. Clinical imaging and postmortem examination suggested multiple large arterial thromboemboli. Histologic examination of vascular lesions revealed markedly thickened tunica intima with polypoid intraluminal projections, which partially to entirely occluded the arterial lumen. The expanded tunica intima was comprised of intimal accumulation of Alcian blue–positive matrix with scattered spindle-to-satellite cells. These cells were positive for von Willebrand factor and vimentin but negative for α–smooth muscle actin, suggesting endothelial origin. Deposition of the intimal mucoid matrix was observed in the elastic and muscular arteries associated with regional ischemic changes. Mucoid emboli, likely from fragmentation of proliferative intimal tissue, were identified in smaller vessels supplied by affected arteries. Based on these findings, we diagnosed systemic mucoid degeneration of the arterial tunica intima. Such systemic arterial degeneration characterized by deposition of mucoid matrix in the tunica intima has not been reported previously in dogs, to our knowledge, and should be distinguished from thromboembolism and other degenerative vascular diseases.
  • Peliosis Hepatis with Chylous Ascites in a Dog
    Kevin Christian Montecillo Gulay; Noriyuki Nagata; Keisuke Aoshima*; Nozomi Shiohara; Atsushi Kobayashi; Mitsuyoshi Takiguchi; Takashi Kimura; *Corresponding author
    Journal of Comparative Pathology, 187, 63, 67, Elsevier {BV}, Aug. 2021, [Peer-reviewed], [Corresponding author], [International Magazine]
    English, Scientific journal, A 6-year-old spayed female Toy Poodle dog was referred to the Hokkaido University Veterinary Teaching Hospital for abdominal distension. Abdominocentesis yielded ascitic fluid that had a mildly increased total protein concentration and a 2.7-fold higher triglyceride concentration than plasma, and was interpreted as chylous ascites. The patient had an enlarged liver, which contained multiple, small, nodular masses and cyst-like structures. Microscopically, these lesions were multifocal dilated spaces containing lymphocytes, endothelial cells, fibrin and islands of hepatocytes. Increased α-smooth muscle actin-positive cells were observed in hepatic sinusoids. Based on these findings, we diagnosed peliosis hepatis with chylous ascites, which is likely to have been due to lymphangiectasia and disrupted hepatic sinusoids. Neither Bartonella spp DNA nor mutations in ACVRL1 and MTM1 genes were detected, although there was a 47-fold increase in hepatic ACVRL1 expression compared with age-matched control liver. To the authors' knowledge, this is the first report of chylous ascites resulting from peliosis hepatis in any species.
  • Duck Tembusu virus induces stronger cellular responses than Japanese encephalitis virus in primary duck neurons and fibroblasts.
    Sittinee Kulprasertsri; Shintaro Kobayashi; Keisuke Aoshima; Atsushi Kobayashi; Takashi Kimura
    Microbiology and immunology, 65, 11, 481, 491, 14 Jul. 2021, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Duck Tembusu virus (DTMUV) and Japanese encephalitis virus (JEV) are mosquito-borne flaviviruses. These two viruses infect ducks; however, they show different neurological outcomes. The mechanism of DTMUV- and JEV-induced neuronal death has not been well investigated. In the present study, we examined the differences in the mechanisms involved in virus-induced cell death and innate immune responses between DTMUV KPS54A61 strain and JEV JaGAr-01 strain using primary duck neurons (DN) and duck fibroblasts (CCL-141). DN and CCL-141 were permissive for the infection and replication of these two viruses, which upregulated the expression of innate immunity genes. Both DTMUV and JEV induced cell death via a caspase-3-dependent manner; however, DTMUV triggered more cell death than JEV did in both CCL-141 and DN. These findings suggest that DTMUV infection causes apoptosis in duck neurons and fibroblasts more strongly than JEV. Levels of the mRNA expression of innate immunity-related genes after DTMUV infection were generally higher than levels after JEV infection, suggesting that DTMUV-induced immune response in duck cells may exhibit toxic effect rather than protective effects. This article is protected by copyright. All rights reserved.
  • Potential for transmission of sporadic Creutzfeldt-Jakob disease through peripheral routes.
    Atsushi Kobayashi; Yoshiko Munesue; Taishi Shimazaki; Keisuke Aoshima; Takashi Kimura; Shirou Mohri; Tetsuyuki Kitamoto
    Laboratory investigation; a journal of technical methods and pathology, 12 Jul. 2021, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Five sporadic Creutzfeldt-Jakob disease (CJD) strains have been identified to date, based on differences in clinicopathological features of the patients, the biochemical properties of abnormal prion proteins, and transmission properties. Recent advances in our knowledge about iatrogenic transmission of sporadic CJD have raised the possibility that the infectivity of sporadic CJD strains through peripheral routes is different from that of intracranial infection. To test this possibility, here we assessed systematically the infectivity of sporadic CJD strains through the peripheral route for the first time using a mouse model expressing human prion protein. Although the infectivity of the V2 and M1 sporadic CJD strains is almost the same in intracerebral transmission studies, the V2 strain infected more efficiently than the M1 strain through the peripheral route. The other sporadic CJD strains examined lacked infectivity. Of note, both the V2 and M1 strains showed preference for mice with the valine homozygosity at the PRNP polymorphic codon. These results indicate that the V2 strain is the most infectious sporadic CJD strain for infection through peripheral routes. In addition, these findings raise the possibility that individuals with the valine homozygosity at the PRNP polymorphic codon might have higher risks of infection through peripheral routes compared with the methionine homozygotes. Thus, preventive measures against the transmission of the V2 sporadic CJD strain will be important for the eradication of iatrogenic CJD transmission through peripheral routes.
  • KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma.
    Kevin Christian Montecillo Gulay; Keisuke Aoshima*; Yuki Shibata; Hironobu Yasui; Qin Yan; Atsushi Kobayashi; Takashi Kimura; *Corresponding author
    Journal of genetics and genomics, 48, 7, 618, 630, Elsevier, 10 Mar. 2021, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we find that lysine-specific demethylase 2b (KDM2B) is highly expressed in HSA cell lines compared with normal canine endothelial cells. Silencing of KDM2B in HSA cells results in increased cell death in vitro compared with the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced KDM2B silencing in tumor xenografts results in decreased tumor sizes compared with the control. Furthermore, KDM2B is also highly expressed in clinical cases of HSA. We hypothesize that pharmacological KDM2B inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treat HSA cells with GSK-J4, a histone demethylase inhibitor, and find that GSK-J4 treatment also induces apoptosis and cell death. In addition, GSK-J4 treatment decreases tumor size. Therefore, we demonstrate that KDM2B acts as an oncogene in HSA by enhancing the DNA damage response. Moreover, we show that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment., 26148785
  • Minocycline prevents primary duck neurons from duck Tembusu virus-induced death
    Sittinee KULPRASERTSRI; Keisuke AOSHIMA; Atsushi KOBAYASHI; Takashi KIMURA
    Journal of Veterinary Medical Science, 83, 4, 734, 741, Japanese Society of Veterinary Science, 2021, [Peer-reviewed]
    Scientific journal
  • Pathological and Immunohistochemical Analyses of Naturally Occurring Equine Glanders Using an Anti-BpaB Antibody
    Ochbayar Erdemsurakh; Baatarjargal Purevdorj; Khurtsbaatar Ochirbat; Altanchimeg Adilbish; Batbaatar Vanaabaatar; Keisuke Aoshima; Atsushi Kobayashi; Takashi Kimura
    Veterinary Pathology, 57, 6, 807, 811, SAGE Publications, Nov. 2020, [Peer-reviewed]
    Scientific journal, Glanders is caused by the gram-negative bacterium Burkholderia mallei. In this study, we investigated the histopathology and immunohistochemical localization of B. mallei in natural cases of equine glanders. Four horses showing clinical signs of nasal discharge and multiple cutaneous nodules or papulae in the hindlimbs and abdomen were reported in Mongolia. They tested positive for B. mallei infection on complement fixation, Rose Bengal agglutination, and mallein tests. Gross and histological lesions observed in these cases were similar to those previously reported in equine glanders. Immunohistochemistry using a monoclonal antibody to B. mallei BpaB showed localization of the bacterial antigen in the cytoplasm of neutrophils, macrophages, epithelioid cells, and multinucleated giant cells in the pyogranulomas and abscesses in target organs. Some alveolar type II cells and bronchiolar epithelial cells also contained the antigen. These results suggest that the anti-BpaB antibody is useful for identifying B. mallei–infected cell types in naturally infected horses.
  • Genome-wide DNA methylation analysis identifies promoter hypermethylation in canine malignant melanoma.
    T Ishizaki; J Yamazaki; J Jelinek; K Aoshima; T Kimura
    Research in veterinary science, 132, 521, 526, Oct. 2020, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Canine malignant melanoma is a common cancer with a high mortality rate. Although previous studies have evaluated various aspects of this tumour, the exact mechanism of tumourigenesis remains unknown. Epigenetic mechanisms, such as DNA methylation, have recently gained attention as aetiological factors for neoplasia in humans. This study aimed to analyse genome-wide DNA methylation patterns in canine malignant melanoma based on next-generation sequencing data. A total of 76,213 CpG sites, including 29,482 sites in CpG islands (CGIs), were analysed using next-generation sequencing of methylation-specific signatures, obtained by sequential digestion with enzymes, to compare normal oral mucosal samples from four healthy dogs, four canine melanoma cell lines (3 oral cavity and 1 skin), and five clinical samples of oral canine melanoma. Malignant melanoma showed increased methylation at thousands of normally unmethylated CpG sites in CGIs and decreased methylation at normally methylated CpG sites in non-CGIs. Interestingly, the promoter regions of 81-393 genes were hypermethylated; 23 of these genes were present in all melanoma cell lines and melanoma clinical samples. Among these 23 genes, six genes with "sequence-specific DNA binding" annotation were significantly enriched, including three Homeobox genes-HMX2, TLX2, and HOXA9-that may be involved in the tumourigenesis of canine malignant melanoma. This study revealed widespread alterations in DNA methylation and a large number of hypermethylated genes in canine malignant melanoma.
  • CECR2 Drives Breast Cancer Metastasis by Suppressing Macrophage Inflammatory Responses
    Meiling Zhang; Zongzhi Z. Liu; Keisuke Aoshima; Yangyi Zhang; Yongyan An; Asako Aoshima; Lok Hei Chan; Sabine M. Lang; Hongyi Sun; Zhenwei Tang; Sara J. Rutter; Carmen J. Booth; Veerle Bossuyt; Xiang Chen; Jon S. Morrow; Lajos Pusztai; David. L. Rimm; Mingzhu Yin; Qin Yan
    bioRxiv, Cold Spring Harbor Laboratory, 10 Sep. 2020, ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, AbstractEpigenetic and transcriptional changes are critical for metastasis, the major cause of cancer-related deaths. Metastatic tumor cells escape immune surveillance more efficiently than tumor cells in the primary sites, but the mechanisms controlling their immune evasion are poorly understood. We found that distal metastases are more immune inert with increased M2 macrophages compared to their matched primary tumors. Acetyl-lysine reader CECR2 is an epigenetic regulator upregulated in metastases and positively associated with M2 macrophages. CECR2 specifically promotes breast cancer metastasis in multiple mouse models, with more profound effect in the immunocompetent setting. Mechanistically, NF-κB family member RELA recruits CECR2 to activate CSF1 and CXCL1, which are critical for macrophage-mediated immunosuppression at the metastatic sites. Furthermore, pharmacological inhibition of CECR2 bromodomain impedes NF-κB-mediated immune suppression by macrophages and inhibits breast cancer metastasis. These results reveal novel therapeutic strategies to treat metastatic breast cancer.

    Statement of SignificanceComparison of matched primary breast tumors and distal metastases show that metastases are more immune inert with increased tumor promoting macrophages. Depletion or pharmacological inhibition of CECR2 inhibits breast cancer metastasis by suppressing macrophage inflammatory responses, nominating CECR2 as a promising therapeutic target for cancer metastasis.

  • Seroprevalence of equine glanders in horses in the central and eastern parts of Mongolia.
    Ochbayar Erdemsurakh; Khurtsbaatar Ochirbat; Ulziisaikhan Gombosuren; Batbold Tserendorj; Baatarjargal Purevdorj; Batbaatar Vanaabaatar; Keisuke Aoshima; Atsushi Kobayashi; Takashi Kimura
    The Journal of veterinary medical science, 07 Jul. 2020, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, Glanders is a contagious and fatal equine disease caused by the gram-negative bacterium Burkholderia mallei.B. mallei is prevalent among horse populations in Asia, the Middle East, and South America. More than four million horses have been registered in Mongolia in 2020. However, the resent prevalence of glanders has not been well investigated. In this study, we aimed to investigate the seropositivity of B. mallei in horse populations in Mongolia using the complement fixation test (CFT) and Rose Bengal plate agglutination test (RBT). We randomly collected blood samples from horses in central and eastern Mongolia between 2018 and 2019. Of 337 horses, 26 (7.7%) and 28 (8.3%) were seropositive using RBT and CFT, respectively. Interestingly, seropositivity in horses resulting from crossbreeding of Mongolian native horses with thoroughbred horses was higher than that in Mongolian native horses. Our observations suggest that equine glanders are still endemic to Mongolia.
  • Long interspersed nucleotide element-1 hypomethylation in canine malignant mucosal melanoma.
    Teita Ishizaki; Jumpei Yamazaki; Shinji Meagawa; Nozomu Yokoyama; Keisuke Aoshima; Mitsuyoshi Takiguchi; Takashi Kimura
    Veterinary and comparative oncology, 18, 4, 854, 860, Wiley, 18 Mar. 2020, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Canine malignant melanoma is a common cancer with a high mortality rate and is a clinically important disease. DNA methylation has been considered to be a potential tumorigenic mechanism through aberrant DNA methylation at promoter region which represses gene transcription. Global hypomethylation could also facilitate chromosome instability. There are few reports regarding DNA methylation in canine malignant melanoma; therefore, the purpose of this study was to examine DNA methylation status of long interspersed nucleotide element-1 (LINE-1) to be a surrogate marker of genome-wide methylation changes in this disease. We measured levels of DNA methylation of two adjacent cytosine-guanine sites on CpG island (CGI) at the putative promoter of canine LINE-1 sequence by bisulphite-pyrosequencing in 41 canine melanoma patient samples as well as six cell lines compared with normal mucosae. The survival rates were obtained from owners or medical records. We found DNA methylation levels of LINE-1 in normal mucosae were methylated. Interestingly, both melanoma cell lines and clinical melanoma samples showed remarkable hypomethylation. In addition, patients with lower LINE-1 methylation showed worse prognosis than those with higher LINE-1 methylation, though the difference did not reach statistical significance (P = .09). Here, we demonstrate that hypomethylation of LINE-1 is an epigenetically aberrant feature in canine melanoma with possible prognostic value.
  • Susceptibility of rat immortalized neuronal cell line Rn33B to equine herpesvirus-1 infection is differentiation-dependent
    Minato E; Kobayashi A; Aoshima K; Fukushi H; Kimura T
    Microbiol Immunol, 64, 2, 123, 132, Wiley, Feb. 2020, [Peer-reviewed]
    English, Scientific journal
  • Acquired resistance to HER2-targeted therapies creates vulnerability to ATP synthase inhibition.
    Gale M; Li Y; Cao J; Liu ZZ; Holmbeck MA; Zhang M; Lang SM; Wu L; Do Carmo M; Gupta S; Aoshima K; DiGiovanna MP; Stern DF; Rimm DL; Shadel GS; Chen X; Yan Q
    Cancer research, Nov. 2019, [Peer-reviewed]
    Scientific journal
  • High drug efflux pump capacity and low DNA damage response induce doxorubicin resistance in canine hemangiosarcoma cell lines.
    Morita A; Aoshima K*; Gulay KCM; Onishi S; Shibata Y; Yasui H; Kobayashi A; Kimura T; *Corresponding author
    Research in veterinary science, 127, 1, 10, Elsevier BV, Oct. 2019, [Peer-reviewed], [Corresponding author]
    Scientific journal
  • Exogenous Expression of Equine MHC Class I Molecules in Mice Increases Susceptibility to Equine Herpesvirus 1 Pulmonary Infection.
    Minato E; Aoshima K; Kobayashi A; Ohnishi N; Sasaki N; Kimura T
    Veterinary pathology, 56, 5, 300985819834616, 710, SAGE Publications, Mar. 2019, [Peer-reviewed]
    Scientific journal, Equine herpesvirus 1 (EHV-1) uses equine major histocompatibility complex class I (MHC class I) as an entry receptor. Exogenous expression of equine MHC class I genes in murine cell lines confers susceptibility to EHV-1 infection. To examine the in vivo role of equine MHC class I as an entry receptor for EHV-1, we generated transgenic (Tg) mice expressing equine MHC class I under the control of the CAG promoter. Equine MHC class I protein was expressed in the liver, spleen, lung, and brain of Tg mice, which was confirmed by Western blot. However, equine MHC class I antigen was only detected in bronchiolar epithelium and not in other tissues, using the immunofluorescence method employed in this study. Both Tg and wild-type (WT) mice developed pneumonia 3 days after intranasal infection with EHV-1. The bronchiolar epithelial cells of Tg mice showed more severe necrosis, compared with those in WT mice. In addition, the number of virus antigen-positive cells in the lungs was higher in Tg mice than in WT mice. These results suggest that exogenous expression of equine MHC class I renders mice more susceptible to EHV-1 infection.
  • A novel combination of prion strain co-occurrence in patients with sporadic Creutzfeldt-Jakob disease.
    Kobayashi A; Iwasaki Y; Takao M; Saito Y; Iwaki T; Qi Z; Torimoto R; Shimazaki T; Munesue Y; Isoda N; Sawa H; Aoshima K; Kimura T; Kondo H; Mohri S; Kitamoto T
    The American journal of pathology, 189, 6, 1276, 1283, Mar. 2019, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Six subgroups of sporadic Creutzfeldt-Jakob disease have been identified by distinctive clinicopathologic features, genotype at polymorphic codon 129 [methionine (M)/valine (V)] of the PRNP gene, and type of abnormal prion proteins (type 1 or 2). In addition to the pure subgroups, mixed neuropathologic features and the coexistence of two types of abnormal prion proteins in the same patient also have been reported. Here, we found that a portion of the patients previously diagnosed as MM1 had neuropathologic characteristics of the MM2 thalamic form (ie, neuronal loss of the inferior olivary nucleus of the medulla). Furthermore, coexistence of biochemical features of the MM2 thalamic form also was confirmed in the identified cases. In addition, in transmission experiments using prion protein-humanized mice, the brain material from the identified case showed weak infectivity and generated characteristic abnormal prion proteins in the inoculated mice resembling those after inoculation with brain material of MM2 thalamic form. Taken together, these results show that the co-occurrence of MM1 and MM2 thalamic form is a novel entity of sporadic Creutzfeldt-Jakob disease prion strain co-occurrence. The present study raises the possibility that the co-occurrence of the MM2 thalamic form might have been overlooked so far because of the scarcity of abnormal prion protein accumulation and restricted neuropathology.
  • Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells
    Keisuke Aoshima*; Yuki Fukui; Kevin Christian Montecillo Gulay; Ochbayar Erdemsurakh; Atsuya Morita; Atsushi Kobayashi; Takashi Kimura; *Corresponding author
    BMC Veterinary Research, 14, 1, 301, Springer Nature America, Inc, Dec. 2018, [Peer-reviewed], [Lead author, Corresponding author]
    Scientific journal
  • Ganglioside synthase knock-out reduces prion disease incubation time in mouse models.
    Kobayashi A; Qi Z; Shimazaki T; Munesue Y; Miyamoto T; Isoda N; Sawa H; Aoshima K; Kimura T; Mohri S; Kitamoto T; Yamashita T; Miyoshi I
    The American journal of pathology, 189, 3, 677, 686, Dec. 2018, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.
  • Anti-arthritic effect of pentosan polysulfate in rats with collagen-induced arthritis
    H.M. Suranji Wijekoon; Sangho Kim; Eugene C. Bwalya; Jing Fang; Keisuke Aoshima; Kenji Hosoya; Masahiro Okumura
    Research in Veterinary Science, 122, 179, 185, Elsevier {BV}, Dec. 2018, [Peer-reviewed], [International Magazine]
    English, Scientific journal, Pentosan polysulfate (PPS) is currently under investigation as a potential disease-modifying antiarthritic agent. In the present study the effects of PPS on arthritic profiles based on clinical score, ankle size, histological changes, and activity of inflammatory mediators using collagen-induced arthritic rat are reported. Model of arthritis was developed in Sprague Dawley rats by intradermal injection of bovine type II collagen emulsified with incomplete Freund's adjuvant. The rats were randomly divided into four groups: normal control, arthritic control, arthritic rats treated with PPS (at dose level 20 μg/g) and arthritic rats treated with meloxicam (2 μg/g). The treatment was continued daily until the day 30. Arthritic biomarkers (cartilage oligomeric matrix protein and tartrate-resistant acid phosphatase 5b) in synovial fluid, expression of inflammatory mediators (interleukin-1β, and tumor necrosis factor-α) and osteoclast marker genes (cathepsin K, tartrate-resistant acid phosphatase) in synovial membrane were measured. Daily administration of PPS to the arthritic rats significantly decreased the severity of arthritis by effectively suppressing the symptoms of arthritis and improving the functional recovery based on clinical score and histopathological evidence. Intriguingly, identical downregulation pattern of arthritis profiles, biological markers as well as relative mRNA levels of osteoclast markers and cytokines were monitored in arthritic rats treated with PPS. In conclusion, PPS exerted protective effects against collagen-induced arthritis in rats. The results suggest that PPS acts as an anti-inflammatory and anti-arthritic agent in decreasing the arthritic effects in collagen-induced arthritic rats.
  • Immune cellular responses to sendai virus infection in D2.B6-Sen1Sen2Sen3 congenic mice, of which three quantitative trait loci responsible for the resistance to infection were introgressed from C57BL/6 mouse into DBA/2 mouse
    Raghda M. F. Abbas; Hassan T. Tag-El-Din-Hassan; Tussapon Boonyarattanasoonthorn; Keisuke Aoshima; Masami Morimatsu; Takashi Agui
    Japanese Journal of Veterinary Research, 66, 2, 93, 103, 01 May 2018, [Peer-reviewed]
    English, Scientific journal
  • Development of a quick bioassay for the evaluation of transmission properties of acquired prion diseases
    Yoshiko Munesue; Taishi Shimazaki; Zechen Qi; Norikazu Isoda; Hirofumi Sawa; Keisuke Aoshima; Takashi Kimura; Shirou Mohri; Tetsuyuki Kitamoto; Atsushi Kobayashi
    Neuroscience Letters, 668, 43, 47, Elsevier Ireland Ltd, 06 Mar. 2018, [Peer-reviewed]
    English, Scientific journal
  • Cellular atypia is negatively correlated with immunohistochemical reactivity of CD31 and vWF expression levels in canine hemangiosarcoma
    Aprilia Maharani; Keisuke Aoshima*; Shinichi Onishi; Kevin Christian Montecillo Gulay; Atsushi Kobayashi; Takashi Kimura. *Corresponding author
    Journal of Veterinary Medical Science, 80, 2, 213, 218, Japanese Society of Veterinary Science, 01 Feb. 2018, [Peer-reviewed], [Corresponding author]
    English, Scientific journal
  • Effects of combretastatin A-4 phosphate on canine normal and tumor tissue derived endothelial cells
    Yusuke Izumi; Keisuke Aoshima; Yuki Hoshino; Satoshi Takagi
    RESEARCH IN VETERINARY SCIENCE, 112, 222, 228, Jun. 2017, [Peer-reviewed]
    English, Scientific journal
  • Use of histone K-M mutants for the analysis of transcriptional regulation in mouse zygotes
    Keisuke Aoshima; Takashi Kimura; Yuki Okada
    Methods in Molecular Biology, 1605, 259, 270, Humana Press Inc., 2017, [Peer-reviewed], [Lead author]
    English, In book
  • KM mutant highlights enhancers in minor ZGA
    Yuki Okada; Keisuke Aoshima
    CELL CYCLE, 14, 16, 2541, 2542, Aug. 2015, [Peer-reviewed]
    English, Scientific journal
  • Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development
    Keisuke Aoshima; Erina Inoue; Hirofumi Sawa; Yuki Okada
    EMBO REPORTS, 16, 7, 803, 812, Jul. 2015, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • Survival of rabid rabbits after intrathecal immunization
    Sawang Kesdangsakonwut; Yuji Sunden; Keisuke Aoshima; Yoshimi Iwaki; Masahiro Okumura; Hirofumi Sawa; Takashi Umemura
    NEUROPATHOLOGY, 34, 3, 277, 283, Jun. 2014, [Peer-reviewed]
    English, Scientific journal
  • Generation of a dual-color reporter mouse line to monitor spermatogenesis in vivo.
    Makino Y; Inoue E; Hada M; Aoshima K; Kitano S; Miyachi H; Okada Y
    Frontiers in cell and developmental biology, 2, 30, 30, 2014, [Peer-reviewed], [International Magazine]
    English, Scientific journal, In vivo fluorescent imaging technique is a strong tool to visualize the various cellular events such as the proliferation, differentiation, migration, and a lineage tracing in living cells requiring no further experimental procedure such as immunostaining. During spermatogenesis, unique and dynamic histone exchanges occur. Since the timing and types of histone exchanges defines the particular stages of spermatogenesis, visualizing certain types of histones in testes is useful not only for researching specific histone dynamics, but also for monitoring the stages of spermatogenesis in vivo. In this study, we report the establishment of two transgenic (Tg) mouse lines expressing histone H4-Venus (H4V) and histone H3.3-mCherry (H33C) fusion proteins in testicular germ cells, and demonstrated their utility for monitoring germ cell development in vivo. Because of the choice of promoter as well as the nature of these histones, H4V and H33C were exclusively expressed in the germ cells of the distinct stages, which allowed the determination of spermatogenic stages in real time. In addition, disappearance of H4V and H33C at particular stages of differentiation/fertilization also represented dynamic histone removal. Collectively, these Tg mice are a valuable resource not only for monitoring differentiation stages, but also for studying the chromatin dynamics of post-natal testicular germ cell development in vivo.
  • Establishment of Alternative Culture Method for Spermatogonial Stem Cells Using Knockout Serum Replacement
    Keisuke Aoshima; Ai Baba; Yoshinori Makino; Yuki Okada
    PLOS ONE, 8, 10, e77715, Oct. 2013, [Peer-reviewed], [Lead author]
    English, Scientific journal
  • [Chromatin dynamics in mouse pronuclear zygotes].
    Keisuke Aoshima
    Seikagaku. The Journal of Japanese Biochemical Society, 85, 4, 278, 283, 4, Apr. 2013, [Lead author]
    Scientific journal
  • Bilateral Segmental Aplasia with Unilateral Uterine Horn Torsion in a Pomeranian Bitch
    Kensuke Nakamura; Masahiro Yamasaki; Tomohiro Osaki; Hiroshi Ohta; Noboru Sasaki; Keisuke Aoshima; Takashi Kimura; Mitsuyoshi Takiguchi
    JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION, 48, 5, 327, 330, Sep. 2012, [Peer-reviewed]
    English, Scientific journal
  • Possible Origin of CSF Antibodies Induced by Intrathecal Immunization and Prophylactic Effects against Intracerebral Rabies Virus Infection
    Keisuke Aoshima; Yuji Sunden; Sachiyo Ishida; Kenji Ochiai; Takashi Umemura
    JOURNAL OF VETERINARY MEDICAL SCIENCE, 73, 10, 1303, 1308, Oct. 2011, [Peer-reviewed], [Lead author]
    English, Scientific journal
■ Other Activities and Achievements
■ Books and other publications
■ Lectures, oral presentations, etc.
  • 〔Major achievements〕Patient-derived models to advance clinically relevant therapy research in canine hemangiosarcoma
    Keisuke Aoshima
    41st World Veterinary Association Congress, 23 Apr. 2026, English, Invited oral presentation
    21 Apr. 2026 - 24 Apr. 2026, [Invited]
  • 〔主要な業績〕血管肉腫研究の進め方―クラウドファンディング活用事例と代謝エピジェネティクス解析
    青島圭佑
    第534回獣医学科セミナー＜外部研究者招聘セミナー＞, 13 Jun. 2025, Japanese, Public discourse
    13 Jun. 2025 - 13 Jun. 2025, 36887866, [Invited]
  • 〔主要な業績〕イヌ血管肉腫の病態解明に向けた次なる研究戦略―患者由来モデルとエピジェネティクス―
    青島圭佑
    第166回 日本獣医学会学術集会, 06 Sep. 2023, Japanese, Invited oral presentation
    05 Sep. 2023 - 07 Sep. 2023, [Invited]
  • 〔Major achievements〕Canine Hemangiosarcoma: A Gateway to Comparative Oncology Advancements Benefiting Both Human and Veterinary Medicine
    Keisuke Aoshima
    International Society of Organoid Research (ISoOR) annual meeting 2023, 08 Aug. 2023, English, Invited oral presentation
    07 Aug. 2023 - 08 Aug. 2023, [Invited]
■ Syllabus
  • 総合獣医療実習, 2024年, 学士課程, 獣医学部
  • 臨床疾病学特論, 2024年, 博士後期課程, 獣医学院
  • 獣医病理学特論, 2024年, 博士後期課程, 獣医学院
  • アカデミックイングリッシュ, 2024年, 博士後期課程, 獣医学院
  • アカデミックイングリッシュ, 2024年, 博士後期課程, 国際感染症学院
  • アドバンスト演習, 2024年, 学士課程, 獣医学部
  • 病理学総論実習, 2024年, 学士課程, 獣医学部
  • 病理学各論実習, 2024年, 学士課程, 獣医学部
■ Affiliated academic society
  • Apr. 2022 - Present
    Japan Cancer Association
  • Japanese college of veterinary pathologists
  • JAPANESE SOCIETY OF VETERINARY SCIENCE
■ Research Themes
  • Redefining Hemangiosarcoma through Lymphangiogenic Factors
    Grants-in-Aid for Scientific Research
    01 Apr. 2025 - 31 Mar. 2028
    青島 圭佑; 平島 一輝
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 25K02164
  • 新規阻害剤から開拓するミトコンドリアによる種間共通の非ATP性がん転移機構の解明
    科学研究費助成事業
    01 Apr. 2025 - 31 Mar. 2028
    平島 一輝; 青島 圭佑
    日本学術振興会, 基盤研究(B), 岐阜大学, 25K02174
  • Elucidation of control mechanisms of viral gene expression in equine herpesvirus type 1-infected neurons
    Grants-in-Aid for Scientific Research
    01 Apr. 2022 - 31 Mar. 2026
    木村 享史; 青島 圭佑
    ウマヘルペスウイルス1型 (EHV-1) は神経細胞へ潜伏感染することが知られており、その成立にはウイルス遺伝子の発現抑制が関与すると推測されるが、詳細は不明である。本研究では、未分化状態においてEHV-1に高い感受性を示すが神経分化によって著しく感受性が低下するラット脳由来細胞株Rn33B-A68B2Mを潜伏感染モデルとして用い、神経分化状態と未分化状態におけるEHV-1増殖性の相違を規定する分子機構を解明し、神経細胞におけるEHV-1潜伏感染の成立機序について考察を行うことを目的とする。
    令和5年度はORF63（ICP0遺伝子）の3'非翻訳領域を認識するmiRNAの同定を試みた。ORF63の読み取り枠直後から3'方向へ向かう193 bpのEHV-1ゲノムDNA断片をpmirGLO miRNA Target Expression Vectorにクローニングし、Rn33B-A68B2M細胞に導入した。EHV-1非感染細胞と感染細胞のそれぞれに対しデュアルルシフェラーゼアッセイを行ったところ、EHV-1感染によりルシフェラーゼ活性の低下がみられたが、これはORF63の3'非翻訳領域をクローニングしていない空ベクターを導入した細胞でも同様にみとめられたため、pmirGLOベクター固有の配列を認識するmiRNAがEHV-1感染時に誘導される可能性が示唆された。また、空ベクターを導入した細胞とORF63の3'非翻訳領域をクローニングしたベクターを導入した細胞とを非感染状態で比較した場合、miRNAの作用を確認するホタルルシフェラーゼのみならず内部標準であるウミシイタケルシフェラーゼについても空ベクター導入細胞の方がより高い活性を示したことから、ORF63の3'非翻訳領域を認識し発現を負に制御する何らかの宿主因子の存在が推測された。また、BALB/cマウスにEHV-1を経鼻接種し、令和4年度の研究において同定されたEHV-1 IE 遺伝子のプロモーター領域より転写されるncRNAの発現をリアルタイムRT-PCRで検索したところ、感染後1日目の肺組織でIE遺伝子と共にncRNAの発現が認められた。
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23K23773
  • ウマヘルペスウイルス1型感染神経細胞におけるウイルス遺伝子発現制御機構の解明
    科学研究費助成事業
    01 Apr. 2022 - 31 Mar. 2026
    木村 享史; 青島 圭佑
    日本学術振興会, 基盤研究(B), 北海道大学, 22H02508
  • Epigenetics for cancer metabolism regulation -elucidation of hemangiosarcoma pathogenesis and search for a novel therapeutic target -
    Grants-in-Aid for Scientific Research
    01 Apr. 2022 - 31 Mar. 2025
    青島 圭佑
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 22K06020
  • Epigenetics of Hemangiosarcoma - The Roles of KDM2B and Hemangiosarcoma Mouse Model -
    Grant-in-Aid for Young Scientists
    Apr. 2020 - Mar. 2022
    Keisuke Aoshima
    We found that KDM2B, a histone demethylase, activated DNA repair system, which allowed tumor cells to avoid apoptosis. We also revealed that this was achieved through histone ubiquitination. Furthermore, KDM2B knockdown and histone demethylase inhibitor GSK-J4 treatment after tumor formation in canine hemangiosarcoma cell line xenograft mouse models decreased and slowed tumor growth, respectively. We also revealed that murine angiosarcoma cell line ISOS-1 derived tumor in BALB/c mice showed similar characteristics to clinical canine hemangiosarcoma cases. ISOS-1 also highly expressed KDM2B that worked in the same mechanism as in canine hemangiosarcoma cell lines.
    Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists, Hokkaido Univerisity, Principal investigator, 20K15654
  • Generating mouse hemangiosarcoma model
    Research grant
    Sep. 2020 - Mar. 2021
    Kuribayashi Scholarship and Academic Foundation., Principal investigator
  • Investigation of the mechanism of equine herpesvirus-1 latency using mice expressing a viral receptor
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    01 Apr. 2016 - 31 Mar. 2020
    Kimura Takashi
    Equine herpesvirus-1 (EHV-1) causes respiratory diseases, abortion and myeloencephalopathy in horses. Little is known about the mechanisms underlying latent EHV-1 infection, due to the lack of small animal models and in vitro infection models. In this study, we developed EHV-1-susceptible cell culture and murine systems by transduction of gene encoding EHV-1 entry receptor, and investigated their suitability as models of EHV-1 latent infection. A rat neuronal cell model showing differentiation dependent resistance to EHV-1 infection may be useful to unveiling the molecular mechanisms of EHV-1 neuropathogenicity and latency.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 16H05022
  • Unveiling the epigenetic mechanism of cellular differentiation in undifferentiated canine hemangiosarcoma cells
    KAKENHI Grant-in-Aid for Young Scientists
    Apr. 2018 - Mar. 2020
    Keisuke AOSHIMA
    We found that histone demethylase KDM2B highly expressed in canine hemangiosarcoma cell lines and clinical samples. KDM2B knockdown halted cell growth and induced apoptosis in vitro. Furthermore, KDM2B knockdown in the tumors inoculated in immunodeficient mice let the tumors started shrinking. Based on these results, we confirmed that KDM2B is responsible for tumor cell growth and survival.
    JSPS, Grant-in-Aid for Early-Career Scientists, Hokkaido University, Principal investigator, Competitive research funding, 18K14575
  • Regulation of abnormal endothelial cells - comparative pathology of hemangiosarcoma and tumor blood vessels -
    Grants-in-Aid for Regional R&D Proposal-Based Program
    Aug. 2016 - Mar. 2017
    Keisuke Aoshima
    Northern Advancement Center for Science & Technology of Hokkaido, Principal investigator, Competitive research funding
  • Analysis of Notch signaling in canine hemangiosarcoma to search for novel treatment targets
    Grant for young researchers
    Jul. 2016 - Mar. 2017
    Keisuke Aoshima
    Akiyama Life Science Foundation, Principal investigator, Competitive research funding
  • Understanding the "genome adaptation" in fertilized embryo
    Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
    01 Apr. 2010 - 31 Mar. 2015
    OKADA Yuki; SHIRAHIGE Katsuhiko; SHIRAHIGE Katsuhiko; MAKINO Katsuhiko; PARK Sung-Joon; AOSHIMA Keisuke; HADA Masashi
    In this study, we aimed i) the establishment of ChIP-seq from very small number of cells, especially mouse preimplantation embyors, and ii) analysis of the (epi)genome reprogramming in fertilized 1-cell embryos using the new ChIP-seq technology.
    In the former project, we obtained substantial improvement of reproducibility in top ~15% sequence reads by modifying the step of linear amplification of sequence libraries, although further modifications are still required to reach the goal. In the latter project, instead of using the new ChIP-seq method, we utilized recently identified histone mutants, which erase endogenous histone methylations at the mutated sites, to investigate the importance of histone methylations in preimplantation embryos. As a result, we identified that paternal-specific H3K4 monomethylation by Mll3/4 is required for transcription of paternal genome in late 1-cell embryos, likely through enhancer activation.
    Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), The University of Tokyo, 22125007
  • Analysis of chromatin dynamics of mouse prenuclear zygote
    Fellowship for Young Scientists, DC2
    Apr. 2013 - Mar. 2015
    Keisuke Aoshima
    本研究はマウス受精卵前核期の雄性前核において大規模に変化するヒストンメチル化修飾の役割とその制御因子を明らかにすることを目的として行われた。その結果，ヒストン H3 の4番目のリジン残基 (H3K4) のメチル化(H3K4me)が初期胚発生に重要であることがわかり，さらにヒストンメチル化酵素であるMixed lineage leukemia 3/4(Mll3/4)が前核期のH3K4meに関与していることが強く示唆された。
    ヒストンのリジン残基をメチオニン残基に変換した変異体(K-M 変異体)は当該部位の内在性メチル化を消去することができる。この変異体を用いて前核期のH3K4me消去を試みたところ，雄性前核においてのみ，H3K4meが消去された。H3K4meは転写活性化のマークとなるヒストン修飾であり，本研究においても前核期におけるH3K4meの消去は受精後に最初に生じる転写活性化minor Zygotic Gene Activation (minor ZGA)を抑制することがわかった。しかし前核期の次の段階，二細胞期に生じる大規模な転写活性化major Zygotic Gene Activation (major ZGA)はH3K4me消去の影響を受けなかった。以上の結果から，雄性前核のおけるH3K4meはminor ZGAの制御に必須であり，minor ZGAで発現する遺伝子群の中に，その後の初期胚発生に必要な遺伝子が含まれていることが示唆された。以上の結果はヒストンメチル化酵素の一つである Mll3/4 をノックダウンした受精卵においても同様に観察されたことから，Mll3/4 が前核期における H3K4meを制御している可能性が示唆された。
    本研究はリプログラミング機構の一端を明らかにし，初期胚発生における遺伝子発現の制御機構に新たな知見をもたらしたと言える。
    Japan Society for the Promotion of Science, 特別研究員奨励費, 北海道大学, Principal investigator, Competitive research funding, 13J01051
  • 海外研修助成
    青島圭佑
    公益財団法人杉野目記念会, Principal investigator, Competitive research funding
■ Industrial Property Rights
  • 抗腫瘍剤
    Patent right, 青島圭佑; 青島克明; 新田直樹; 舟津充洋, 国立大学法人北海道大学, 株式会社アスコ
    特願2026-031731, 27 Feb. 2026