National Health Research Institutes

7 ARTICLES PUBLISHED IN JoVE

JoVE Journal

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Chun A. Changou^1,2, Deanna L. Wolfson², Balpreet Singh Ahluwalia^2,3, Richard J. Bold^4,5, Hsing-Jien Kung^5,6, Frank Y.S. Chuang^1,2,5

¹Department of Biochemistry and Molecular Medicine, University of California, Davis , ²NSF Center for Biophotonics Science & Technology, University of California, Davis , ³University of Tromsø, ⁴Department of Surgery (Division of Surgical Oncology), University of California, Davis , ⁵UC Davis Comprehensive Cancer Center, University of California, Davis , ⁶Department of Biological Chemistry, University of California, Davis

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Bioengineering

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Ching-Hui Lin^1,2, Hao-Chen Chang^1,2, Chia-Hsien Hsu^1,2,3

¹Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Taiwan, ²Tissue Engineering and Regenerative Medicine, National Chung Hsing University, ³Institute of NanoEngineering and MicroSystems, National Tsing Hua University

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Bioengineering

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Yee Fun Lee¹, Chou-Ching Lin², Jung-Sung Cheng¹, Gin-Shin Chen¹

¹Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, ²Department of Neurology, National Cheng Kung University Hospital

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Developmental Biology

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Meng-Chieh Lin^*1, Shih-Yin Chen^*1, Pei-Lin He¹, Wen-Ting Luo¹, Hua-Jung Li¹

¹Institute of Cellular and System Medicine, National Health Research Institutes

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

Biology

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Wan-Shan Yang¹, Mel Campbell², Hsing-Jien Kung^2,3,4,5, Pei-Ching Chang^1,6

¹Institute of Microbiology and Immunology, National Yang-Ming University, ²UC Davis Cancer Center, University of California, Davis, ³Department of Biochemistry and Molecular Medicine, University of California, Davis, ⁴Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, ⁵Division of Molecular and Genomic Medicine, National Health Research Institutes, ⁶Center for Infectious Disease and Cancer Research, Kaohsiung Medical University

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Bioengineering

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cheng-Kun He^1,2, Ya-Wen Chen³, Ssu-Han Wang³, Chia-Hsien Hsu^1,2

¹Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, ²Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, ³National Institute of Cancer Research, National Health Research Institutes

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Biology

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Cai-Ying Yang^*1,2, Jia-Horung Hung^*3, Chi-Jung Wu^4,5, Zhao-Xiang Wang^1,2, Shih-Han Wang⁶, Hao-Chun Liaw², I-Huang Lin³, Chun-Keung Yu^1,6, Tak-Wah Wong^2,7,8

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.