Latent pH-responsive ratiometric fluorescent cluster based on self-assembled photoactivated SNARF derivatives

We have developed a self-assembled fluorescent cluster comprising a seminaphthorhodafluor (SNARF) derivative protected by a photoremovable o-nitrobenzyl group. Prior to UV irradiation, a colorless and nonfluorescent cluster was spontaneously assembled in aqueous solution. After UV irradiation, the self-assembled cluster remained intact and showed a large enhancement in pH-responsive fluorescence. The unique pH responsive fluorescent cluster could be used as a dual-emissive ratiometric fluorescent pH probe not only in the test tube but also in HeLa cell cultures

テイサンソ ヒョウテキ ヤクザイ ノ メディシナル ブリコラージュ ト ジセダイ イヤクヒン ボロン トレースドラッグ ノ ソウセイ

Hypoxia is now considered a fundamentally important characteristic of the tumor microenvironment. A discovery of the hypoxia inducible factor（HIF）has led to a rapidly increasing understanding of the molecular mechanisms involved in tumor hypoxia. This in turn has led to the current extensive interest in the signal molecules related to tumor hypoxia as potential molecular targets for cancer therapeutics. In this paper we give a medicinal bricolage overview of recent advances in hypoxia-targeting drugs research. These hypoxia-targeting drugs include antiangiogenic- and sugar-hybrid-hypoxic cell radiosensitizers and hypoxic cytotoxins, hypoxia-targeting boron neutron capture therapy（BNCT）drugs. The evaluation of ADME-tox and pharmacokinetic properties of drugs are extremely important and essential in their discovery process and their lifetime. Traditionally, as well known traceable drugs, their radiolabeled compounds have been studied for their purposes. However, there are some inherent problems such as their half-life and regulation of experimental facilities. For the purpose of overcoming these problems and creating drugs with functions required for systems biology or emerging physiology, we designed, as a traceable nextgeneration drug model, wholly innovative drugs named“boron tracedrug,”their architecture of which were embedded boron atom in their scaffold or skeleton. These boron tracedrugs could be detected whenever and wherever you need to access in their lifetime. We called them“honnête homme”drugs. Also utilizing this specific property of boron tracedrugs, we suggested the neutron dynamic therapy（NDT）

改良型pＨプロ－ブによる細胞内pHの定量的な計測方法の開発

SNARF is one of the most commonly used pH indicators for biological applications, owing to its unique fluorescent characteristics. For intracellular applications, esterase-substrate derivatives of SNARF, such as acetate or acetoxymethyl esters, have been employed previously as a generally accepted strategy to increase cell permeability. Unfortunately such cell-permeable SNARF derivatives retain significant fluorescence in aqueous solution, a property which results in a low signal-to-noise ratio. This in turn can lead to incorrect intracellullar pH measurements. Here we describe UTX-40, a newly designed SNARF derivative that successfully addresses these problems. In aqueous solution, UTX-40 is devoid of fluorescence before ester hydrolysis because it exists in aqueous environment as nano-scaled aggregates. As UTX-40 is converted into SNARF by enzymatic hydrolysis inside the cell, the aggregates become diffused and monomeric SNARF displays its characteristic fluorescent properties. The results of our studies reported in this communication demonstrate clearly the benefits of UTX-40 as an intracellular pH indicator. Since intracellular localized fluorescence was observed without cell-washing, the efficient uptake of intracellular fluorescence was confirmed. In addition, the actual intracellular pH and changes in intracellular pH caused by drug addition were monitored. The low background noise produced by UTX-40 is a property of this new pH probe that should be particularly advantageous for in vivo usage because, for this application, it is difficult to wash out the redundant probe

低酸素細胞特異的な蛍光イメージング剤の開発

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wavelength absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts. UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with reduced background signal

Simple method for large-scale production of macrophage activating factor GcMAF

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing

ジェネティック制御下にある発育鶏卵を用いた工学的in vivo 薬剤評価系の開発

The drug discovery research of clinical-use antioxidants, which may control various vascular disorders caused by the oxidative stress, is extremely important. We present the development of an in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryonic chorioallantoic membrane (CAM). In the case of 12-days chick embryos, the topical administration of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) induced their vascular injuries against the CAM veins and venous capillaries but without substantial fatal damage. Artepillin C, a potent natural antioxidant, did not show the chick embryo’s venous injury, and pre-treatment with artepillin C would tend to protect the CAM veins injuries induced by the post-administration of AAPH. These results suggest that artepillin C might be able to protect the AAPH-induced vascular injury in in vivo. In conclusion, we showed the possibility of in vivo evaluation system of antioxidants for their vascular protective activities using the chick embryo

Association Between SLFN11 and Antitumor Activity of Trabectedin

Background/Aim: Trabectedin is a DNA-damaging agent and has been approved for the treatment of patients with advanced soft tissue sarcoma. Schlafen 11 (SLFN11) was identified as a dominant determinant of the response to DNA-damaging agents. The aim of the study was to clarify the association between SLFN11 expression and the antitumor activity of trabectedin. Materials and Methods: The antitumor activity of trabectedin was evaluated under different expression levels of SLFN11 regulated by RNA interference and CRISPR-Cas9 systems, and the combined antitumor activity of ataxia telangiectasia and Rad3-related protein kinase (ATR) inhibitor and trabectedin in sarcoma cell lines using in vitro a cell viability assay and in vivo xenograft models. Results: SLFN11-knockdown cell lines had a lower sensitivity to trabectedin, compared to parental cells. ATR inhibitor enhanced the antitumor activity of trabectedin in SLFN11-knockdown cells and in a SLFN11-knockout xenograft model. Conclusion: SLFN11 expression might be a key factor in the antitumor activity of trabectedin

Time- and Learner-Dependent Hidden Markov Model for Writing Process Analysis Using Keystroke Log Data

Teaching writing strategies based on writing processes has attracted wide attention as a method for developing writing skills. The writing process can be generally defined as a sequence of subtasks, such as planning, formulation, and revision. Therefore, instructor feedback is often given based on sequence patterns of those subtasks. For such feedback, instructors need to analyze sequence patterns for all learners, which becomes problematic as the number of learners increases. To resolve this problem, this study proposes a new machine-learning method that estimates sequence patterns from keystroke log data. Specifically, we propose an extension of the Gaussian hidden Markov model that incorporates parameters representing temporal change in a subtask appearance distribution for each learner. Furthermore, we propose a collapsed Gibbs sampling algorithm as the parameter estimation method for the proposed model. We demonstrate effectiveness of the proposed model by applying it to actual keystroke log datasets

青色光は滑膜肉腫に対して活性酸素種によるミトコンドリア機能障害を起こし、アポトーシスとオートファジーを誘導する

Background: Synovial sarcoma (SS) has limited treatment options and there is an urgent need to develop a novel therapeutic strategy to treat SS. Blue light (BL) has been shown to inhibit the growth of several cancer cells. However, the efficacy of BL in soft tissue sarcomas such as SS has not been demonstrated, and the detailed mechanism underlying the antitumor activity of BL is not fully understood. In this study, we investigated the antitumor effect of BL on SS. Methods: Human SS cell lines were continuously irradiated with BL using light-emitting diodes (LEDs) in an incubator for in vitro analysis. The chicken chorioallantoic membrane (CAM) tumors and xenograft tumors in mice were subjected to daily BL irradiation with LEDs. Results: BL caused growth inhibition of SS cells and histological changes in CAM tumors. BL also suppressed the migration and invasion abilities of SS cells. The type of cell death in SS cells was revealed to be apoptosis. Furthermore, BL induced excessive production of reactive oxygen species (ROS) in mitochondria, resulting in oxidative stress and malfunctioned mitochondria. Reducing the production of ROS using N-acetylcysteine (NAC), a ROS scavenger, attenuated the inhibitory effect of BL on SS cells and mitochondrial dysfunction. In addition, BL induced autophagy, which was suppressed by the administration of NAC. The autophagy inhibitor of 3-methyladenine and small interfering RNA against the autophagy marker light chain 3B facilitated apoptotic cell death. Moreover, BL suppressed tumor growth in a mouse xenograft model. Conclusion: Taken together, our results revealed that BL induced apoptosis via the ROS-mitochondrial signaling pathway, and autophagy was activated in response to the production of ROS, which protected SS cells from apoptosis. Therefore, BL is a promising candidate for the development of an antitumor therapeutic strategy targeting SS