ゲノム編集技術を用いた脳の進化発生学研究の展開

京都府立医科大学大学院神経発生生物学京都府立医科大学医学部医学科近年急速に進歩したCRISPR/Cas9 によるゲノム編集技術は、これまでのマウスES細胞を用いた相同遺伝子組換えによる標的遺伝子破壊の手法に大きな変革をもたらした。本稿では、この技術を用いて種特異的な遺伝子機能の解析を行った最近の研究について概説するとともに、我々が行ったニワトリ胚におけるPax6 遺伝子の標的遺伝子破壊の研究について紹介する。特に、CRISPR/Cas9 を用いた組織特異的遺伝子破壊に必要な検証実験について考察し、脳の進化発生学研究におけるあらたな研究戦略について議論する

Functional Analysis of MeCP2 Mutations Associated with Rett Syndrome Using Transient Expression Systems

レット症候群は生後半年から1歳半ころに発症する重度の精神発達遅滞を伴う疾患で女児の1万人から1万5千人に1人に発症する頻度の高い遺伝子疾患である。この疾患の原因遺伝子が最近MeCP2遺伝子であることが判明した。レット症候群の患者でみられる変異がMeCP2の本来の機能にどのような影響を及ぼすかを理解することは、レット症候群の病態を解明する上での手がかりになる。MeCP2は2つの機能ドメインを持ち、一つはメチル化CpGに結合するメチル化結合ドメイン(MBD)で、もう一つはヒストン脱アセチル化酵素をリクルートするSin3Aと結合する転写抑制ドメイン(TRD)である。報告されている変異の中でミスセンス変異の多くは、この二つのドメイン内でみられ、特にMBD内での変異の割合は多い。MBD内のミスセンス変異のMeCP2機能への影響を把握するため、培養細胞を用いた遺伝子導入発現系を開発して解析を行った

Human Hepatocyte Growth Factor Promotes Functional Recovery in Primates after Spinal Cord Injury

Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery. In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI

Functional Characterisation of MeCP2 Mutatiions Found in Male Patients with X Linked Mental Retardation

MeCP2の遺伝子変異は、Rett症候群以外の疾患の患者でも見つかり、X染色体性の精神発達遅滞を伴う男性患者においても報告された。これらの患者ではMBD内の変異として137番目のGluからGlyと140番目AlaからValのアミノ酸変異が確認された。これらの変異に関して、開発した二つの機能解析系を用いて解析を行ったところ、140番目の変異では、メチル化DNAに対しての転写抑制活性は完全に維持されており、137番目の変異ではわずかに転写抑制活性の低下がみられる程度であった。また、マウス細胞のヘテロクロマチン親和性についても140番と137番目の変異は共に明らかな点状の像を示し、親和性は維持されていた。これらの遺伝性の精神発達遅滞を伴う男性患者でのMeCP2の変異は、その機能への影響がレット症候群の場合と比較して軽度であるため、Rett症候群とは異なる病態を呈する成因となっている可能性が示唆された

Inactivation of the Influenza Virus by a Supplemental Fermented Plant Product (Manda Koso)

Manda Koso is a commercial fermented plant product (FPP) made from 53 types of fruits and vegetables that are fermented for more than 3 years. We hypothesized that the FPP can prevent infection by influenza virus and human norovirus. Therefore, we investigated the effects of the FPP on influenza virus and feline calicivirus, a surrogate of human norovirus. We found that 10% FPP inactivated the influenza virus but not the feline calicivirus. Inhibition of the influenza virus was highly concentration-dependent: 1% and 0.3% FPP showed reduced inactivation efficacy. The effects of the FPP on the influenza virus-infected cells were investigated by addition of the FPP to the culture medium after virus infection. No suppressive effect of the FPP on influenza replication in MDCK cells was observed. The results showed that the FPP could inactivate influenza virus by affecting the virus particles

Development of a fluoride-responsive amide bond cleavage device that is potentially applicable to a traceable linker

A fluoride-responsive (FR) amino acid that induces amide bond cleavage upon the addition of a fluoride was developed, and it was applied to a FR traceable linker. By the use of an alkyne-containing peptide as a model of an alkynylatd target protein of a bioactive compound, introduction of the FR traceable linker onto the peptide was achieved. Subsequent fluoride induced cleavage of the linker followed by labeling of the released peptide derivative was also conducted to examine the potential applicability of the FR traceable linker to the enrichment and labeling of alkynylated target molecules

Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins

A traceable linker that is potentially applicable to identification of a target protein of bioactive compounds was developed. It enabled not only thiol-induced cleavage of the linker for enrichment of the target protein but also selective labelling to pick out the target from contaminated non-target proteins for facile identification

Immunological profile in a family with nephrogenic diabetes insipidus with a novel 11 kb deletion in AVPR2 and ARHGAP4 genes

<p>Abstract</p> <p>Background</p> <p>Congenital nephrogenic diabetes insipidus (NDI) is characterised by an inability to concentrate urine despite normal or elevated plasma levels of the antidiuretic hormone arginine vasopressin. We report a Japanese extended family with NDI caused by an 11.2-kb deletion that includes the entire <it>AVPR2 </it>locus and approximately half of the <it>Rho GTPase-activating protein 4 </it>(<it>ARHGAP4</it>) locus. ARHGAP4 belongs to the RhoGAP family, Rho GTPases are critical regulators of many cellular activities, such as motility and proliferation which enhances intrinsic GTPase activity.</p> <p>ARHGAP4 is expressed at high levels in hematopoietic cells, and it has been reported that an NDI patient lacking <it>AVPR2 </it>and all of <it>ARHGAP4 </it>showed immunodeficiency characterised by a marked reduction in the number of circulating CD3+ cells and almost complete absence of CD8+ cells.</p> <p>Methods</p> <p>PCR and sequencing were performed to identify the deleted region in the Japanese NDI patients. Immunological profiles of the NDI patients were analysed by flow cytometry. We also investigated the gene expression profiles of peripheral blood mononuclear cells (PBMC) from NDI patients and healthy controls in microarray technique.</p> <p>Results</p> <p>We evaluated subjects (one child and two adults) with 11.2-kb deletion that includes the entire <it>AVPR2 </it>locus and approximately half of the <it>ARHGAP4</it>. Hematologic tests showed a reduction of CD4+ cells in one adult patient, a reduction in CD8+ cells in the paediatric patient, and a slight reduction in the serum IgG levels in the adult patients, but none of them showed susceptibility to infection. Gene expression profiling of PBMC lacking <it>ARHGAP4 </it>revealed that expression of RhoGAP family genes was not influenced greatly by the lack of <it>ARHGAP4</it>.</p> <p>Conclusion</p> <p>These results suggest that loss of <it>ARHGAP4 </it>expression is not compensated for by other family members. ARHGAP4 may play some role in lymphocyte differentiation but partial loss of <it>ARHGAP4 </it>does not result in clinical immunodeficiency.</p

Protocol for a multicentre, prospective observational study of elective neck dissection for clinically node-negative oral tongue squamous cell carcinoma (END-TC study)

Introduction: In early-stage oral tongue squamous cell carcinoma (OTSCC), elective neck dissection (END) is recommended when occult lymph node metastasis is suspected; however, there is no unanimous consensus on the risks and benefits of END in such cases. The management of clinically node-negative (cN0) OTSCC remains controversial. This study, therefore, aimed to evaluate the efficacy of END and its impact on the quality of life (QoL) of patients with cN0 OTSCC. Methods and analysis: This is a prospective, multicentre, nonrandomised observational study. The choice of whether to perform END at the same time as resection of the primary tumour is based on institutional policy and patient preference. The primary endpoint of this study is 3-year overall survival. The secondary endpoint are 3-year disease-specific survival, 3-year relapse-free survival and the impact on patient QoL. Propensity score-matching analysis will be performed to reduce selection bias. Ethics and dissemination: This study was approved by the Clinical Research Review Board of the Nagasaki University. The protocol of this study was registered at the University Hospital Medical Information Network Clinical Trials Registry. The datasets generated during the current study will be available from the corresponding author on reasonable request. The results will be disseminated internationally, through scientific and professional conferences and in peer-reviewed medical journals