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The Hospital for Sick Children Research Institute

5 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Electroporation of Functional Bacterial Effectors into Mammalian Cells
Ryan L. Sontag 1, Cosmin Mihai 2, Galya Orr 2, Alexei Savchenko 3, Tatiana Skarina 3, Hong Cui 3, John R. Cort 1, Joshua N. Adkins 1, Roslyn N. Brown 4
1Biological Sciences Division, Pacific Northwest National Laboratory, 2Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, 3Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto, 4Center for Bioproducts and Bioenergy, Washington State University

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

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Developmental Biology

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach
Yun-Ui Bae 1, Hoon-Ki Sung 2,3, Jae-Ryong Kim 1
1Department of Biochemistry and Molecular Biology and Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, 2Physiology and Experimental Medicine Program, The Hospital for Sick Children Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

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JoVE Journal

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
June Ereño-Orbea *1, Taylor Sicard *1,2, Hong Cui 1, Indira Akula 1, Jean-Philippe Julien 1,2,3
1Program in Molecular Medicine, The Hospital for Sick Children Research Institute, 2Department of Biochemistry, University of Toronto, 3Department of Immunology, University of Toronto

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

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Biology

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining
Yanqing Jiang *1, Joanna Lan-Hing Yeung *1, Ju Hee Lee 1,2, James An 1, Patrick E. Steadman 3, Jae-Ryong Kim 4, Hoon-Ki Sung 1,2,5
1Translational Medicine Program, The Hospital for Sick Children, 2Department of Laboratory Medicine and Pathobiology, University of Toronto, 3Neurosciences & Mental Health Program, The Hospital for Sick Children, 4Department of Biochemistry and Molecular Biology, Smart-Aging Convergence Research Center, College of Medicine, Yeungnam University, 5Banting and Best Diabetes Centre, University of Toronto

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

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Biology

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice
Ri Youn Kim *1,2, Ju Hee Lee *3,4, Yena Oh 1,2, Hoon-Ki Sung 3,4,5, Kyoung-Han Kim 1,2
1University of Ottawa Heart Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Translational Medicine Program, The Hospital for Sick Children, 4Department of Laboratory Medicine and Pathobiology, University of Toronto, 5Banting and Best Diabetes Centre, University of Toronto

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

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