Shen Y.,University of Alberta |
Chen Y.,University of Alberta |
Wu J.,University of Alberta |
Shaner N.C.,Scintillon Institute |
Campbell R.E.,University of Alberta
PLoS ONE | Year: 2017
MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (ëex = 460 nm and ëem = 610 nm) and a red-shifted (RDS)-mCherry variant (ëex = 600 nm and ëem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry's fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging. © 2017 Shen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Buxbaum J.N.,Scripps Research Institute |
Buxbaum J.N.,Scintillon Institute |
Johansson J.,Karolinska Institutet
Frontiers in Neuroscience | Year: 2017
Amyloid fibrils are physiologically insoluble biophysically specific β-sheet rich structures formed by the aggregation of misfolded proteins. In vivo tissue amyloid formation is responsible for more than 30 different disease states in humans and other mammals. One of these, Alzheimer's disease (AD), is the most common form of human dementia for which there is currently no definitive treatment. Amyloid fibril formation by the amyloid β-peptide (Aβ) is considered to be an underlying cause of AD, and strategies designed to reduce Aβ production and/or its toxic effects are being extensively investigated in both laboratory and clinical settings. Transthyretin (TTR) and proteins containing a BRICHOS domain are etiologically associated with specific amyloid diseases in the CNS and other organs. Nonetheless, it has been observed that TTR and BRICHOS structures are efficient inhibitors of Aβ fibril formation and toxicity in vitro and in vivo, raising the possibility that some amyloidogenic proteins, or their precursors, possess properties that may be harnessed for combating AD and other amyloidoses. Herein, we review properties of TTR and the BRICHOS domain and discuss how their abilities to interfere with amyloid formation may be employed in the development of novel treatments for AD. © 2017 Buxbaum and Johansson.
Nakamura T.,Scintillon Institute |
Lipton S.A.,Scintillon Institute |
Lipton S.A.,University of California at San Diego
Trends in Pharmacological Sciences | Year: 2016
At physiological levels, nitric oxide (NO) contributes to the maintenance of normal neuronal activity and survival, thus serving as an important regulatory mechanism in the central nervous system. By contrast, accumulating evidence suggests that exposure to environmental toxins or the normal aging process can trigger excessive production of reactive oxygen/nitrogen species (such as NO), contributing to the etiology of several neurodegenerative diseases. We highlight here protein S-nitrosylation, resulting from covalent attachment of an NO group to a cysteine thiol of the target protein, as a ubiquitous effector of NO signaling in both health and disease. We review our current understanding of this redox-dependent post-translational modification under neurodegenerative conditions, and evaluate how targeting dysregulated protein S-nitrosylation can lead to novel therapeutics. © 2015 Elsevier Ltd. All rights reserved.
Nolan J.P.,Scintillon Institute |
Jones J.C.,U.S. National Institutes of Health
Platelets | Year: 2017
The composition and function of platelet-derived extracellular vesicles (EVs) in health and in disease are a major topic of investigation in biomedical research. However, efforts to delineate specific molecular repertoires and roles for different types of EVs in the circulation are limited not only by the lack of flow cytometers capable of analyzing submicron- and nano-materials across the full size spectrum of plasma EVs, but also by the lack of standardized methods and reference materials that would permit inter-laboratory reproducibility for these analyses. In this review, we summarize the flow cytometry of EVs, with a focus on platelet vesicles in plasma. In addition to delineating the basic principles that govern what precautions must be considered when using flow cytometry for the analysis of platelet vesicles, we provide an overview for how to standardize, control, annotate, and report EV flow cytometry data reproducibly, while looking forward to a next generation of high sensitivity instruments for the analysis of EVs and other submicron biomaterials in the circulation. © 2017 Taylor & Francis.
Zhu S.,Xiamen University |
Ma L.,Xiamen University |
Wang S.,Xiamen University |
Chen C.,Xiamen University |
And 6 more authors.
ACS Nano | Year: 2014
Ultrasensitive detection and characterization of single nanoparticles (<100 nm) is important in nanotechnology and life sciences. Direct measurement of the elastically scattered light from individual nanoparticles represents the simplest and the most direct method for particle detection. However, the sixth-power dependence of scattering intensity on particle size renders very small particles indistinguishable from the background. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, here we report the development of high sensitivity flow cytometry (HSFCM) that achieves real-time light-scattering detection of single silica and gold nanoparticles as small as 24 and 7 nm in diameter, respectively. This unprecedented sensitivity enables high-resolution sizing of single nanoparticles directly based on their scattered intensity. With a resolution comparable to that of TEM and the ease and speed of flow cytometric analysis, HSFCM is particularly suitable for nanoparticle size distribution analysis of polydisperse/heterogeneous/mixed samples. Through concurrent fluorescence detection, simultaneous insights into the size and payload variations of engineered nanoparticles are demonstrated with two forms of clinical nanomedicine. By offering quantitative multiparameter analysis of single nanoparticles in liquid suspensions at a throughput of up to 10 000 particles per minute, HSFCM represents a major advance both in light-scattering detection technology and in nanoparticle characterization. © 2014 American Chemical Society.
Shaner N.C.,Scintillon Institute |
Shaner N.C.,Allele Biotechnologyandpharmaceuticals Inc |
Lambert G.G.,Scintillon Institute |
Chammas A.,Allele Biotechnologyandpharmaceuticals Inc |
And 10 more authors.
Nature Methods | Year: 2013
We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins. © 2013 Nature America, Inc. All rights reserved.
Wang G.,Boston Childrens Hospital |
McCain M.L.,Wyss Institute for Biologically Inspired Engineering |
Yang L.,Harvard University |
He A.,Boston Childrens Hospital |
And 28 more authors.
Nature Medicine | Year: 2014
Study of monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combined patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene encoding tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS 'heart-on-chip' tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole-cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies and advances iPSC-based in vitro modeling of cardiomyopathy. © 2014 Nature America, Inc. All rights reserved.
Shaner N.C.,Scintillon Institute
Methods in Cell Biology | Year: 2014
More than 20 years after their discovery, fluorescent proteins (FPs) continue to be the subject of massive engineering efforts yielding continued improvements. Among these efforts are many aspects that should be of great interest to quantitative imaging users. With new variants frequently introduced into the research community, "tried and true" FPs that have been relied on for many years may now be due for upgrades to more modern variants. However, the dizzying array of FPs now available can make the initial act of narrowing down the potential choices an intimidating prospect. This chapter describes the FP properties that most strongly impact their performance in quantitative imaging experiments, along with their physical origins as they are currently understood. A workflow for evaluating a given FP in the researcher's chosen experimental system (e.g., a specific cell line) is described. © 2014 Elsevier Inc.
Nolan J.P.,Scintillon Institute
Current Protocols in Cytometry | Year: 2015
Evidence suggests that extracellular vesicles (EVs) can play roles in physiology and pathology, providing impetus to explore their use as diagnostic and therapeutic targets. However, EVs are also small, heterogeneous, and difficult to measure, and so this potential has not yet been realized. The development of improved approaches to EV detection and characterization will be critical to further understanding their roles in physiology and disease. Flow cytometry has been a popular tool for measuring cell-derived EVs, but has often been used in an uncritical manner in which fundamental principles and limitations of the instrument are ignored. Recent efforts to standardize procedures and document the effects of different methodologies have helped to address this shortcoming, butmuchwork remains. In this paper, I address some of the instrument, reagent, and analysis considerations relevant to measurement of individual EVs in flow, with the aim of clarifying a path to quantitative and standardized measurement of these interesting and potentially important biological nanoparticles. © 2015 by John Wiley & Sons, Inc.
PubMed | Scintillon Institute and University of California at San Diego
Type: Journal Article | Journal: Trends in pharmacological sciences | Year: 2016
At physiological levels, nitric oxide (NO) contributes to the maintenance of normal neuronal activity and survival, thus serving as an important regulatory mechanism in the central nervous system. By contrast, accumulating evidence suggests that exposure to environmental toxins or the normal aging process can trigger excessive production of reactive oxygen/nitrogen species (such as NO), contributing to the etiology of several neurodegenerative diseases. We highlight here protein S-nitrosylation, resulting from covalent attachment of an NO group to a cysteine thiol of the target protein, as a ubiquitous effector of NO signaling in both health and disease. We review our current understanding of this redox-dependent post-translational modification under neurodegenerative conditions, and evaluate how targeting dysregulated protein S-nitrosylation can lead to novel therapeutics.