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The Hospital for Sick Children Research Institute

5 ARTICLES PUBLISHED IN JoVE

Immunology and Infection

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Ryan L. Sontag¹, Cosmin Mihai², Galya Orr², Alexei Savchenko³, Tatiana Skarina³, Hong Cui³, John R. Cort¹, Joshua N. Adkins¹, Roslyn N. Brown⁴

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.

Developmental Biology

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Yun-Ui Bae¹, Hoon-Ki Sung^2,3, Jae-Ryong Kim¹

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.

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 Cui¹, Indira Akula¹, 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.

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 An¹, Patrick E. Steadman³, Jae-Ryong Kim⁴, 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.

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.