Newcastle University View Institution's Website 19 articles published in JoVE Biology Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis Cassie R. Bakshani1,2, Jiraporn Sangta3, Sarana Sommano3, William G. T. Willats1 1Department of Biology, School of Natural and Environmental Sciences, Newcastle University, 2Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, 3Department of Plant and Soil Sciences, Faculty of Agriculture, Chiang Mai University Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples. Biology Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels Siji Kavil*1, Alex Laverick*1, Colette J. Whitfield1, Alice M. Banks2, Thomas P. Howard1 1School of Natural and Environmental Sciences, Newcastle University, 2Department of Life Sciences, Imperial College London Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase. Cancer Research A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma Marco Y. W. Zaki*1,2, Shishir Shetty*2, Alex L. Wilkinson2, Daniel A. Patten2, Fiona Oakley*3, Helen Reeves*4,5 1Department of Biochemistry, Faculty of Pharmacy, Minia University, 2National Institute for Health Research Birmingham Liver Biomedical Research Unit and Centre for Liver and Gastrointestinal Research, Institute of Immunology and Immunotherapy, University of Birmingham, 3Newcastle Fibrosis Research Group, Biosciences Institute, Faculty of Medical Sciences, Newcastle University, 4Newcastle University Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, 5The Liver Unit, Department of Medicine, Freeman Hospital, Newcastle-upon-Tyne Hospitals NHS Foundation Trust Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma. Cancer Research Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma Hiroki Kobayashi1,2,3,4, Krystyna A. Gieniec1,2, Jia Q. Ng1,2, Jarrad Goyne1,2, Tamsin R. M. Lannagan1,2, Elaine M. Thomas1,2, Georgette Radford1,2, Tongtong Wang1,2, Nobumi Suzuki1,2,5, Mari Ichinose1,2, Josephine A. Wright2, Laura Vrbanac1,2, Alastair D. Burt6, Masahide Takahashi3,4,7, Atsushi Enomoto3, Daniel L. Worthley2, Susan L. Woods1,2 1Adelaide Medical School, University of Adelaide, 2South Australian Health and Medical Research Institute (SAHMRI), 3Department of Pathology, Nagoya University Graduate School of Medicine, 4Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 5Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, 6Translational and Clinical Research Institute, Newcastle University, 7International Center for Cell and Gene Therapy, Fujita Health University Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies. Biochemistry Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells Vinciane Saint-Criq1, Iram J. Haq2,3, Aaron I. Gardner2, James P. Garnett2,4, Christopher Ward1,2, Malcolm Brodlie2,3, Michael A. Gray1 1Epithelial Research Group, Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, 2Respiratory Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, 3Paediatric Respiratory Medicine, Great North Children's Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust, 4Boehringer Ingelheim Pharma GmbH & Co We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader. Environment A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci Simon Blaser1,2,3, Hanspeter Diem4, Andreas von Felten4, Morgan Gueuning1, Michael Andreou5, Neil Boonham6,7, Jennifer Tomlinson6, Pie Müller2,3, Jürg Utzinger2,3, Beatrice Frey1, Jürg E. Frey1, Andreas Bühlmann8 1Department of Method Development and Analytics, Agroscope, 2Swiss Tropical and Public Health Institute, 3University of Basel, 4Swiss Federal Plant Protection Service, Federal Office for Agriculture, 5OptiGene Limited, 6Fera Science Limited, 7School of Natural and Environmental Sciences, Newcastle University, 8Department of Plants and Plant Products, Agroscope This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports. Behavior A Real-world What-Where-When Memory Test Tom V. Smulders1, Amber Black-Dominique2, Tahsina S. Choudhury3, Simona E. Constantinescu3, Kyriaki Foka3, Tom J. Walker3, Kevin Dick4, Stephen Bradwel4, R. Hamish McAllister-Williams3, Peter Gallagher3 1Institute of Neuroscience and Centre for Behavior & Evolution, Newcastle University, 2School of Psychology, Newcastle University, 3Institute of Neuroscience, Newcastle University, 4Digital Media Services, Newcastle University The Real-World What-Where-When memory test is a novel episodic memory test, in which participants need to recall which objects have been hidden in which locations on which of two distinct occasions. It is easy to run and is sensitive to normal cognitive aging. Biochemistry High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits Julia Schückel*1, Stjepan Krešimir Kračun*1, William G. T. Willats2 1Department for Plant and Environmental Sciences, University of Copenhagen, 2School of Agriculture, Food and Rural Development, Newcastle University A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases. Neuroscience A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris) Carolyn Ma1, Sébastien Kessler1, Alexander Simpson1, Geraldine Wright1 1Institute of Neuroscience, Newcastle University A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution. Behavior The Emotional Stroop Task: Assessing Cognitive Performance under Exposure to Emotional Content Moshe Shay Ben-Haim1, Paul Williams2, Zachary Howard2, Yaniv Mama3, Ami Eidels2, Daniel Algom1 1School of Psychological Sciences, Tel-Aviv University, 2School of Psychology, University of Newcastle, 3Department of Behavioral Sciences, Ariel University The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than those of neutral words. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them. Developmental Biology Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover Gavin D. Richardson1 1Institute of Genetic Medicine, International Centre for Life, Newcastle University Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy. Medicine Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome Alicja M. Baczynska1,2, Sarah Shaw3, Helen C. Roberts1,2,3,5, Cyrus Cooper2,3,4, Avan Aihie Sayer1,2,3,5,6, Harnish P. Patel1,2,3 1Academic Geriatric Medicine, University of Southampton, University Hospital Southampton, 2National Institute for Health Research Southampton Biomedical Research Center, University of Southampton and University Hospital Southampton NHS Foundation Trust, 3MRC Lifecourse Epidemiology Unit, University of Southampton, 4National Institute for Health Research Musculoskeletal Biomedical Research Unit, University of Oxford, 5National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care, 6Newcastle University Institute of Ageing and Institute of Health and Society, Newcastle University This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome. Developmental Biology Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients Simon W. Fellgett1, Simon A. Ramsbottom2, Richard J. Maguire3, Stephen Cross4, Peter O'Toole5, Mary E. Pownall5 1Department of Biomedical Science, The Bateson Centre, University of Sheffield, 2Institute of Genetic Medicine, Newcastle University, 3Department of Cardiovascular Science, The Bateson Centre, University of Sheffield, 4School of Biochemistry, University of Bristol, 5Biology Department, University of York The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients. Bioengineering Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms Derek S. Samarian1, Nicholas S. Jakubovics2, Ting L. Luo1, Alexander H. Rickard1 1Department of Epidemiology, School of Public Health, The University of Michigan, 2Centre for Oral Health Research, School of Dental Sciences, Newcastle University The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque. Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted. Neuroscience Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo Simon Tual-Chalot1, Kathleen R. Allinson1, Marcus Fruttiger2, Helen M. Arthur1 1Institute of Genetic Medicine, Newcastle University, 2UCL Institute of Ophthalmology, University College, London The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies. Biology A Quantitative Fitness Analysis Workflow A.P. Banks*1, C. Lawless*1, D.A. Lydall1 1Institute for Cell and Molecular Biosciences, Newcastle University Medical School Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed. Biology Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos Maureen Sinclair1, Jun-Yong Huang1 1Institute for Cell and Molecular Biosciences, University of Newcastle, United Kingdom The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system. Neuroscience Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes Wolfgang Stein1, Carola Städele1, Peter Andras2 1Institute of Neurobiology, Ulm University, 2School of Computing Science & Institute of Neuroscience, Newcastle University Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion. Biology Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures Maya Sieber-Blum1,2, Yaofei Hu2 1Institute of Human Genetics and Northeast England Stem Cell Institute, Newcastle University, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.