David Geffen School of Medicine

8 ARTICLES PUBLISHED IN JoVE

Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

Farid Rahimi¹, Panchanan Maiti¹, Gal Bitan^2,3

¹Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, ²Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, ³Department of Neurology, University of California, Los Angeles

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

Neuroscience

Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease

Farid Rahimi¹, Gal Bitan^1,2,3

¹Department of Neurology, David Geffen School of Medicine, ²Molecular Biology Institute, University of California, Los Angeles, ³Brain Research Institute, University of California, Los Angeles

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.

Genetics

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Mithun Mitra^1,2, Ha Neul Lee^1,2,3, Hilary A. Coller^1,2,3

¹Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, ²Department of Biological Chemistry, David Geffen School of Medicine, ³Molecular Biology Institute Interdepartmental Program, University of California, Los Angeles

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Biochemistry

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

David Jelinek^1,2, Aimee Flores^1,3, Melanie Uebelhoer¹, Vincent Pasque², Kathrin Plath^2,3, M. Luisa Iruela-Arispe^1,3, Heather R. Christofk^2,3, William E. Lowry^1,3, Hilary A. Coller^1,2,3

¹Department of Molecular, Cell and Developmental Biology, UCLA, ²Department of Biological Chemistry, David Geffen School of Medicine, ³Molecular Biology Institute Interdepartmental Program, UCLA

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Chemistry

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

Eric Schopf^*1, Christopher M. Waldmann^*2,3, Jeffrey Collins^2,4, Christopher Drake¹, Roger Slavik^2,3, R. Michael van Dam^2,4

¹SOFIE, ²Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles (UCLA), ³Ahmanson Translational Imaging Division, University of California, Los Angeles (UCLA), ⁴Crump Institute for Molecular Imaging, University of California, Los Angeles (UCLA)

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [¹⁸F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

Cancer Research

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

David Jelinek^1,2, Ellen Ran Zhang^1,2,3, Aaron Ambrus^1,2, Erin Haley³, Emily Guinn^1,2, Austin Vo^1,2, Peter Le^1,2, Ayse Elif Kesaf^1,2, Jennifer Nguyen^1,2, Lily Guo^1,2, Destiny Frederick^1,2, Zhengyang Sun^1,2, Natalie Guo³, Parker Sevier^1,2, Eric Bilotta^1,2, Kaiser Atai^1,2,4, Laurent Voisin^1,2, Hilary A. Coller^1,2,4

¹Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, ²Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, ³Department of Molecular Biology, Princeton University, ⁴Molecular Biology Institute, University of California, Los Angeles

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Chemistry

Optimization of Radiochemical Reactions using Droplet Arrays

Alejandra Rios^1,2, Travis S. Holloway^2,3, Jia Wang^2,4, R. Michael van Dam^1,2,3,4

¹Physics and Biology in Medicine Interdepartmental Graduate Program, University of California Los Angeles (UCLA), ²Crump Institute of Molecular Imaging, UCLA, ³Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, ⁴Department of Bioengineering, UCLA

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

Bioengineering

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Nicholas Kluge¹, Shyue-An Chan¹, Jeffrey L. Ardell^2,3, Corey Smith¹

¹Department of Physiology and Biophysics, Case Western Reserve University, ²University of California Los Angeles (UCLA) Cardiac Arrhythmia Center, David Geffen School of Medicine, ³UCLA Neurocardiology Research Program of Excellence, UCLA

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.