Trinity College Dublin

9 ARTICLES PUBLISHED IN JoVE

Biology

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

¹Membrane Structural and Functional Biology Group, Schools of Biochemistry and Immunology and Medicine, Trinity College Dublin

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Biology

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Dianfan Li¹, Coilín Boland¹, Kilian Walsh¹, Martin Caffrey¹

¹Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry & Immunology, Trinity College Dublin

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Biology

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Dianfan Li¹, Coilín Boland¹, David Aragao¹, Kilian Walsh¹, Martin Caffrey¹

¹Membrane Structural and Functional Biology Group, Schools of Medicine and Biochemistry & Immunology, Trinity College Dublin

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Bioengineering

Harmonic Nanoparticles for Regenerative Research

Flavio Ronzoni¹, Thibaud Magouroux², Remi Vernet¹, Jérôme Extermann³, Darragh Crotty⁴, Adriele Prina-Mello⁵, Daniel Ciepielewski⁶, Yuri Volkov⁴, Luigi Bonacina², Jean-Pierre Wolf², Marisa Jaconi¹

¹Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, ²Physics Department, GAP-Biophotonics, University of Geneva, ³Laboratoire d'Optique Biomédicale (LOB), Faculté des Sciences et Techniques de l'Ingénieur, École Polytechnique Fédérale de Lausanne, ⁴Department of Clinical Medicine, School of Medicine, Trinity College Dublin, ⁵School of Medicine and CRANN, Trinity College Dublin, ⁶Nikon AG Instruments

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Medicine

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Natalia Muñoz-Wolf^1,2, Analía Rial¹, José M. Saavedra¹, José A. Chabalgoity¹

¹Departamento de Desarrollo Biotecnológico, Universidad de la República, ²Present Affiliation: Trinity Biomedical Science Institute, Trinity College Dublin

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Engineering

Preparation of Liquid-exfoliated Transition Metal Dichalcogenide Nanosheets with Controlled Size and Thickness: A State of the Art Protocol

Claudia Backes¹, Damien Hanlon², Beata M. Szydlowska², Andrew Harvey², Ronan J. Smith², Thomas M. Higgins¹, Jonathan N. Coleman²

¹Chair of Applied Physical Chemistry, Ruprecht-Karls University Heidelberg, ²School of Physics and CRANN, Trinity College Dublin

A protocol for the liquid exfoliation of layered materials to nanosheets, their size selection and size measurement by microscopic and spectroscopic techniques is presented.

Developmental Biology

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

Balta Lucendo-Villarin¹, Hassan Rashidi^1,2, Sharmin Alhaque^1,3, Lena Fischer^1,4, Jose Meseguer-Ripolles¹, Yu Wang¹, Cliona O'Farrelly⁴, Michael Themis³, David C. Hay¹

¹MRC Centre for Regenerative Medicine, University of Edinburgh, ²UCL Great Ormond Street Institute of Child Health, University College London, ³Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, ⁴School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

Environment

Sampling, Identification and Characterization of Microplastics Release from Polypropylene Baby Feeding Bottle during Daily Use

Dunzhu Li^*1,2, Luming Yang^*1,2, Rachel Kavanagh¹, Liwen Xiao^2,3, Yunhong Shi^1,2, Daniel K. Kehoe¹, Emmet D. Sheerin^1,4, Yurii K. Gun’ko^4,5, John J. Boland^1,4, Jing Jing Wang¹

¹AMBER Research Centre and Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN), Trinity College Dublin, ²Department of Civil, Structural and Environmental Engineering, Trinity College Dublin, ³TrinityHaus, Trinity College Dublin, ⁴School of Chemistry, Trinity College Dublin, ⁵BEACON, Bioeconomy SFI Research Centre, University College Dublin

This study detailed a reliable and cost-effective protocol for microplastics collection and detection from the daily use of plastic products.

Medicine

Delineating the Metabolic Phenotype of Biopsy-Derived Kidney Cells

Masataka Kawamura^1,2,3, Catherine Parmentier^1,3, Lisa A. Robinson^2,4,5, Markus Selzner^1,3, Caitriona M. McEvoy^6,7,8, Julia M. Murphy^1,6,9, Maya G. Bozzo-Rey¹⁰, Sarah Q. Crome^1,6,9, Ana Konvalinka^1,4,6,11,12, Sergi Clotet-Freixas¹⁰

¹Soham and Shaila Ajmera Family Transplant Centre, University Health Network, ²Division of Nephrology, The Hospital for Sick Children, ³Division of General Surgery, University Health Network, ⁴Institute of Medical Science, University of Toronto, ⁵Program in Cell Biology, The Hospital for Sick Children Research Institute, ⁶Toronto General Hospital Research Institute, University Health Network, ⁷Division of Nephrology, Tallaght University Hospital, ⁸Trinity Kidney Centre, Trinity College Dublin, ⁹Department of Immunology, University of Toronto, ¹⁰Division of Nephrology, McMaster University and St. Joseph's Healthcare, ¹¹Division of Nephrology, Department of Medicine, University Health Network, ¹²Department of Laboratory Medicine and Pathobiology, University of Toronto

This protocol describes a unique method to conduct metabolic profiling of biopsy-derived kidney cells. The approach presented was optimized in healthy adult male pigs and has the potential to enhance the real-time assessment of kidney allograft viability. This method may also help uncover new biology across multiple metabolism-based kidney diseases.