Japan Science and Technology Agency (JST), PRESTO 6 articles published in JoVE Biochemistry Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device Masatoshi Maeki1,2, Yuto Okada3, Shuya Uno3, Ayuka Niwa3, Akihiko Ishida1, Hirofumi Tani1, Manabu Tokeshi1 1Division of Applied Chemistry, Faculty of Engineering, Hokkaido University, 2JST PRESTO, 3Graduate School of Chemical Sciences and Engineering, Hokkaido University Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP. Behavior Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration Kazuki Shigyou*1, Haruka Maeoka*1, Ryuji Igarashi2,3,4, Takuma Sugi1 1Program of Biomedical Science, Graduate School of Integrated Sciences for Life, Hiroshima University, 2Institute for Quantum Life Science, National Institute for Quantum and Radiological Science and Technology, 3National Institute for Radiological Sciences, National Institute for Quantum and Radiological Science and Technology, 4JST, PRESTO Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations. Biochemistry Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level Simon Leclerc1,2, Youri Arntz2, Yuichi Taniguchi1,3 1Laboratory for Cell Systems Control, RIKEN Center for Biosystems Dynamics Research, RIKEN, 2Laboratoire de Biomatériaux et Bioimagerie, INSERM, 3PRESTO, Japan Science and Technology Agency Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level. Neuroscience Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators Hiroko Bannai1,2, Matsumi Hirose2, Fumihiro Niwa2,3, Katsuhiko Mikoshiba2 1Japan Science and Technology Agency, PRESTO, 2Laboratory for Developmental Neurobiology, RIKEN Center for Brain Science, 3École Normale Supèrieure, Institut de Biologie de l'ENS (IBENS), Institut national de la santè et de la recherche mèdicale (INSERM), Centre national de la recherche scientifique (CNRS), École Normale Supèrieure, PSL Research University Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators. Engineering A Polymer-based Piezoelectric Vibration Energy Harvester with a 3D Meshed-Core Structure Takuya Tsukamoto1, Yohei Umino1, Kotaro Hashikura1, Sachie Shiomi1, Kou Yamada1, Takaaki Suzuki1,2 1Gunma University, 2JST PRESTO In this study, we fabricated a flexible 3D mesh structure and applied it to the elastic layer of a bimorph cantilever-type vibration energy harvester for the purpose of lowering resonance frequency and increasing output power. Biology A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging Shigeyuki Betsuyaku1,2,3,4, Nobuhiko Nomura3,4, Hiroo Fukuda2 1Japan Science and Technology Agency (JST), PRESTO, 2Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 3Faculty of Life and Environmental Sciences, University of Tsukuba, 4Microbiology Research Center for Sustainability, University of Tsukuba We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.
