ホヤ幼生におけるGnRH神経系とグリア上衣細胞の機能解析

ゴナドトロピン放出ホルモン（GnRH）は、下垂体からゴナドトロピンを放出させる神経ペプチドであり、脊椎動物の生殖機能の重要な調節因子である。また、GnRHは神経修飾因子として脳の各所や嗅上皮などの活動の調節を行っていると考えられている。以前の研究で、カタユウレイボヤ幼生において、GnRHをコードするgnrh2遺伝子が脊椎動物の後脳・脊髄の相同器官である運動神経節と神経索で発現し、その受容体が筋肉細胞で発現していることが明らかにされた。ホヤ幼生は生殖活動とは無関係な発生段階であるため、GnRHの新奇生理機能が推測された。本研究では、ホヤ幼生におけるGnRHおよびgnrh2発現細胞の生理機能を明らかにすることを目的として、gnrh2を発現する細胞を同定し、蛍光Ca2+センサータンパク質を用いて、これらの細胞の活動をリアルタイムで可視化した。その結果、(1)gnrh2を発現する特定のコリン作動性ニューロンが尾芽胚期に周期的な活動を示す、(2)gnrh2を発現する尾部神経索のグリア上衣細胞で幼生の遊泳運動や眼点への光刺激と連動したCa2+濃度変化が起こる、という２つの新しい現象を発見した。運動に伴うグリア細胞の活動は過去に知られていない現象であるため、この現象のしくみと生理機能を明らかにすることを目的として、光活性化イオンチャネルを用いた光遺伝学的手法とカルシウムイメージングを併用し、運動神経節のニューロンの活動とグリア上衣細胞の活動の相関性を解析した。その結果、コリン作動性ニューロンを活性化した後、グリア上衣細胞内のCa2+濃度が上昇した。また、単一細胞トランスクリプトーム解析の結果、ニコチン性アセチルコリン受容体をはじめとする複数種類の神経伝達物質受容体がグリア上衣細胞に発現していることが明らかになった。これらの結果より、ホヤ幼生において、コリン作動性ニューロンからの情報をグリア上衣細胞が受け取り、相互作用していることが示唆された。以上、本研究では、gnrh2発現細胞の解析を通して、ホヤ幼生のいくつかのニューロンのユニークな性状を明らかにするとともに、グリア細胞の活動およびニューロンとの相互作用に関する新たな知見を得た。脊椎動物においても、グリア細胞がニューロンと情報交換を行い、脳機能に重要な役割をもつことが明らかになりつつあるが、詳細は未解明である。本研究では、ホヤ幼生において、ニューロンだけでなく、グリア細胞が神経や筋肉の活動の制御に能動的に関与している可能性が示され、グリア細胞の機能と役割の理解に貢献することが期待される。甲南大学令和4年度(2022年度

Percutaneous cryoablation for clinical T3a renal cell carcinoma (< 7 cm) with segmental vein involvement or perinephric fat invasion based on preoperative evaluation of high-resolution multidetector computed tomography scan

Purpose　To retrospectively assess the feasibility, safety, renal function, technique efficacy rate, and survival of patients with clinical T3a renal cell carcinoma (RCC). Materials and methods　Sixteen cryoablation sessions were performed in 14 patients (10 men; mean age, 69.8 ± 10.5 years; range, 49–90 years) with 14 clear cell T3a RCCs (mean, 3.3 ± 0.9 cm; range, 1.9–5.2 cm). One patient was on dialysis. Transcatheter arterial embolization was performed before cryoablation in 15 sessions. The primary endpoint was the technique efficacy rate. The secondary endpoints included feasibility, safety, renal function, and survival. Results　Cryoablation was technically successful in all RCC cases. In two RCCs, cryoablation was performed twice because of local tumor progression. No major adverse events were observed. All patients were alive without metastases, with a median follow-up of 45 months (6−93 months). Complete response was achieved by cryoablation in 11 RCCs (78.6%). The primary and secondary technique efficacy rates were 77.1% and 84.4% at 1 year, 57.9% and 73.9% at 3 years, and 57.9% and 73.9% at 5 years, respectively. One patient underwent dialysis given a total contralateral nephrectomy due to another RCC 1 month after initial cryoablation and a total ipsilateral nephrectomy 46 months after initial cryoablation due to local progression. Except for two dialysis patients, of the 12 patients with a median follow-up of 41 months (6–93 months), none were on dialysis. Conclusion　Cryoablation was safe and effective in T3a RCC, which mainly involved the renal venous branches and may represent an alternative treatment for inoperable patients

A novel histone exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A–H2B

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes

Evolution of Developmental Programs for the Midline Structures in Chordates: Insights From Gene Regulation in the Floor Plate and Hypochord Homologues of Ciona Embryos

In vertebrate embryos, dorsal midline tissues, including the notochord, the prechordal plate, and the floor plate, play important roles in patterning of the central nervous system, somites, and endodermal tissues by producing extracellular signaling molecules, such as Sonic hedgehog (Shh). In Ciona, hedgehog.b, one of the two hedgehog genes, is expressed in the floor plate of the embryonic neural tube, while none of the hedgehog genes are expressed in the notochord. We have identified a cis-regulatory region of hedgehog.b that was sufficient to drive a reporter gene expression in the floor plate. The hedgehog.b cis-regulatory region also drove ectopic expression of the reporter gene in the endodermal strand, suggesting that the floor plate and the endodermal strand share a part of their gene regulatory programs. The endodermal strand occupies the same topographic position of the embryo as does the vertebrate hypochord, which consists of a row of single cells lined up immediately ventral to the notochord. The hypochord shares expression of several genes with the floor plate, including Shh and FoxA, and play a role in dorsal aorta development. Whole-embryo single-cell transcriptome analysis identified a number of genes specifically expressed in both the floor plate and the endodermal strand in Ciona tailbud embryos. A Ciona FoxA ortholog FoxA.a is shown to be a candidate transcriptional activator for the midline gene battery. The present findings suggest an ancient evolutionary origin of a common developmental program for the midline structures in Olfactores

Percutaneous cryoablation for clinical T3a renal cell carcinoma (< 7 cm) with segmental vein involvement or perinephric fat invasion based on preoperative evaluation of high-resolution multidetector computed tomography scan

Purpose　To retrospectively assess the feasibility, safety, renal function, technique efficacy rate, and survival of patients with clinical T3a renal cell carcinoma (RCC). Materials and methods　Sixteen cryoablation sessions were performed in 14 patients (10 men; mean age, 69.8 ± 10.5 years; range, 49–90 years) with 14 clear cell T3a RCCs (mean, 3.3 ± 0.9 cm; range, 1.9–5.2 cm). One patient was on dialysis. Transcatheter arterial embolization was performed before cryoablation in 15 sessions. The primary endpoint was the technique efficacy rate. The secondary endpoints included feasibility, safety, renal function, and survival. Results　Cryoablation was technically successful in all RCC cases. In two RCCs, cryoablation was performed twice because of local tumor progression. No major adverse events were observed. All patients were alive without metastases, with a median follow-up of 45 months (6−93 months). Complete response was achieved by cryoablation in 11 RCCs (78.6%). The primary and secondary technique efficacy rates were 77.1% and 84.4% at 1 year, 57.9% and 73.9% at 3 years, and 57.9% and 73.9% at 5 years, respectively. One patient underwent dialysis given a total contralateral nephrectomy due to another RCC 1 month after initial cryoablation and a total ipsilateral nephrectomy 46 months after initial cryoablation due to local progression. Except for two dialysis patients, of the 12 patients with a median follow-up of 41 months (6–93 months), none were on dialysis. Conclusion　Cryoablation was safe and effective in T3a RCC, which mainly involved the renal venous branches and may represent an alternative treatment for inoperable patients