Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury.

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Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury.

Nat Commun. 2019 01 31;10(1):518

Authors: Bellver-Landete V, Bretheau F, Mailhot B, Vallières N, Lessard M, Janelle ME, Vernoux N, Tremblay MÈ, Fuehrmann T, Shoichet MS, Lacroix S

Abstract
The role of microglia in spinal cord injury (SCI) remains poorly understood and is often confused with the response of macrophages. Here, we use specific transgenic mouse lines and depleting agents to understand the response of microglia after SCI. We find that microglia are highly dynamic and proliferate extensively during the first two weeks, accumulating around the lesion. There, activated microglia position themselves at the interface between infiltrating leukocytes and astrocytes, which proliferate and form a scar in response to microglia-derived factors, such as IGF-1. Depletion of microglia after SCI causes disruption of glial scar formation, enhances parenchymal immune infiltrates, reduces neuronal and oligodendrocyte survival, and impairs locomotor recovery. Conversely, increased microglial proliferation, induced by local M-CSF delivery, reduces lesion size and enhances functional recovery. Altogether, our results identify microglia as a key cellular component of the scar that develops after SCI to protect neural tissue.

PMID: 30705270 [PubMed - indexed for MEDLINE]



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ARGLU1 is a transcriptional coactivator and splicing regulator important for stress hormone signaling and development.

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ARGLU1 is a transcriptional coactivator and splicing regulator important for stress hormone signaling and development.

Nucleic Acids Res. 2019 Jan 30;:

Authors: Magomedova L, Tiefenbach J, Zilberman E, Le Billan F, Voisin V, Saikali M, Boivin V, Robitaille M, Gueroussov S, Irimia M, Ray D, Patel R, Xu C, Jeyasuria P, Bader GD, Hughes TR, Morris QD, Scott MS, Krause H, Angers S, Blencowe BJ, Cummins CL

Abstract
Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.

PMID: 30698747 [PubMed - as supplied by publisher]



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A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations.

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A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations.

Sci Rep. 2019 Jan 29;9(1):842

Authors: Nixon AML, Duque A, Yelle N, McLaughlin M, Davoudi S, Pedley NM, Haynes J, Brown KR, Pan J, Hart T, Gilbert PM, Singh SK, O'Brien CA, Sidhu SS, Moffat J

Abstract
Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin β6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin β6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.

PMID: 30696911 [PubMed - in process]



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Local delivery of stabilized chondroitinase abc degrades chondroitin sulfate proteoglycans in stroke-injured rat brains.

Local delivery of stabilized chondroitinase abc degrades chondroitin sulfate proteoglycans in stroke-injured rat brains.

J Control Release. 2019 Jan 25;:

Authors: Hettiaratchi MH, O'Meara MJ, Teal CJ, Payne SL, Pickering AJ, Shoichet MS

Abstract
Central nervous system (CNS) injuries, such as stroke and spinal cord injuries, result in the formation of a proteoglycan-rich glial scar, which acts as a barrier to axonal regrowth and limits the regenerative capacity of the CNS. Chondroitinase ABC (ChABC) is a potent bacterial enzyme that degrades the chondroitin sulfate proteoglycan (CSPG) component of the glial scar and promotes tissue recovery; however, its use is significantly limited by its inherent instability at physiological temperatures. Here, we demonstrate that ChABC can be stabilized using site-directed mutagenesis and covalent modification with poly(ethylene glycol) chains (i.e. PEGylation). Rosetta protein structure modeling was used to screen >20,000 single point mutations, and four potentially stabilizing mutations were tested in vitro. One of the mutations, N1000G (asparagine ➔ glycine at residue 1000), significantly improved the long-term activity of the protein, doubling its functional half-life. PEGylation of this ChABC mutant inhibited unfolding and aggregation and resulted in prolonged bioactivity with a 10-fold increase in activity compared to the unmodified protein after two days. Local, affinity-controlled release of the modified protein (PEG-N1000G-ChABC) was achieved by expressing it as a fusion protein with Src homology 3 (SH3) and delivering the protein from a methylcellulose hydrogel modified with SH3 binding peptides. This affinity-based release strategy provided sustained PEG-N1000G-ChABC-SH3 release over several days in vitro. Direct implantation of the hydrogel delivery vehicle containing stabilized PEG-N1000G-ChABC-SH3 onto the rat brain cortex in a sub-acute model of stroke resulted in significantly reduced CSPG levels in the penumbra of 49% at 14 and 40% at 28 days post-injury compared to animals treated with the vehicle alone.

PMID: 30690102 [PubMed - as supplied by publisher]



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"Plug-n-Play" Sensing with Digital Microfluidics.

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"Plug-n-Play" Sensing with Digital Microfluidics.

Anal Chem. 2019 Jan 24;:

Authors: de Campos RPS, Rackus DG, Shih R, Zhao C, Liu X, Wheeler AR

Abstract
Digital microfluidics (DMF) is a platform that enables highly reconfigurable and automated fluidic operations using a generic device architecture. A unique hallmark of DMF is its "flexibility": a generic device design can be used and reused for many different, divergent fluidic operations. The flexibility of DMF is compromised when devices are permanently modified with embedded sensors. Here we introduce a solution to the "flexibility gap" between fluidic operations in digital microfluidics and embedded sensors: "plug-n-play DMF" (PnP-DMF). In PnP-DMF, devices are designed to allow for rapid and seamless exchange of sensors depending on the application needs. This paper provides "proof of concept" for PnP-DMF using commercial biosensors for glucose and β-ketone, a custom paper-based electrochemical sensor for lactate, and a generic screen-printed electroanalytical cell. We demonstrate that hot-swapping sensors between experiments allows for convenient implementation of complex processes such as automated analysis of blood samples by standard addition. Finally, we explored the suitability for using PnP sensors in tandem with other sensing modalities, combining biosensor-based electrochemical measurement of glucose with a chemiluminescent magnetic bead-based sandwich immunoassay for insulin. The latter is notable, as it constitutes the first report of an analysis of different analytes in both the supernatant and precipitate from a single sample-aliquot in a microfluidic device. The results presented here highlight the versatility of PnP-DMF, illustrating how it may be useful for a wide range of applications in diagnostics and beyond.

PMID: 30676737 [PubMed - as supplied by publisher]



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Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap.

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Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap.

Nat Protoc. 2019 02;14(2):482-517

Authors: Reimand J, Isserlin R, Voisin V, Kucera M, Tannus-Lopes C, Rostamianfar A, Wadi L, Meyer M, Wong J, Xu C, Merico D, Bader GD

Abstract
Pathway enrichment analysis helps researchers gain mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. We explain the procedures of pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome-sequencing experiments. The protocol comprises three major steps: definition of a gene list from omics data, determination of statistically enriched pathways, and visualization and interpretation of the results. We describe how to use this protocol with published examples of differentially expressed genes and mutated cancer genes; however, the principles can be applied to diverse types of omics data. The protocol describes innovative visualization techniques, provides comprehensive background and troubleshooting guidelines, and uses freely available and frequently updated software, including g:Profiler, Gene Set Enrichment Analysis (GSEA), Cytoscape and EnrichmentMap. The complete protocol can be performed in ~4.5 h and is designed for use by biologists with no prior bioinformatics training.

PMID: 30664679 [PubMed - indexed for MEDLINE]



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A web application and service for imputing and visualizing missense variant effect maps.

A web application and service for imputing and visualizing missense variant effect maps.

Bioinformatics. 2019 Jan 14;:

Authors: Yingzhou W, Weile J, Cote A, Sun S, Knapp J, Verby M, Roth FP

Abstract
Motivation: The promise of personalized genomic medicine depends on our ability to assess the functional impact of rare sequence variation. Multiplexed assays of variant effect experimentally measure the functional impact of missense variants on a massive scale. However, even after such an assay, many missense variants remain poorly measured. Here we describe a software pipeline and application to impute missing information in experimentally determined variant effect maps.
Availability: http://impute.varianteffect.org source code: https://github.com/joewuca/imputation.
Supplementary information: Supplementary data are available at Bioinformatics online.

PMID: 30649215 [PubMed - as supplied by publisher]



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Digital microfluidics and nuclear magnetic resonance spectroscopy for in situ diffusion measurements and reaction monitoring.

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Digital microfluidics and nuclear magnetic resonance spectroscopy for in situ diffusion measurements and reaction monitoring.

Lab Chip. 2019 Jan 16;:

Authors: Swyer I, von der Ecken S, Wu B, Jenne A, Soong R, Vincent F, Schmidig D, Frei T, Busse F, Stronks HJ, Simpson AJ, Wheeler AR

Abstract
In recent years microcoils and related structures have been developed to increase the mass sensitivity of nuclear magnetic resonance spectroscopy, allowing this extremely powerful analytical technique to be extended to small sample volumes (<5 μl). In general, microchannels have been used to deliver the samples of interest to these microcoils; however, these systems tend to have large dead volumes and require more complex fluidic connections. Here, we introduce a two-plate digital microfluidic (DMF) strategy to interface small-volume samples with NMR microcoils. In this system, a planar microcoil is surrounded by a copper plane that serves as the counter-electrode for the digital microfluidic device, allowing for precise control of droplet position and shape. This feature allows for the user-determination of the orientation of droplets relative to the main axes of the shim stack, permitting improved shimming and a more homogeneous magnetic field inside the droplet below the microcoil, which leads to improved spectral lineshape. This, along with high-fidelity droplet actuation, allows for rapid shimming strategies (developed over decades for vertically oriented NMR tubes) to be employed, permitting the determination of reaction-product diffusion coefficients as well as quantitative monitoring of reactive intermediates. We propose that this system paves the way for new and exciting applications for in situ analysis of small samples by NMR spectroscopy.

PMID: 30648175 [PubMed - as supplied by publisher]



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Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments.

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Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments.

Nat Protoc. 2019 02;14(2):415-440

Authors: Simpkins SW, Deshpande R, Nelson J, Li SC, Piotrowski JS, Ward HN, Yashiroda Y, Osada H, Yoshida M, Boone C, Myers CL

Abstract
The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical-genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis. The software has also successfully processed data from Schizosaccharomyces pombe, Escherichia coli, and Zymomonas mobilis mutant collections. We provide general recommendations for the design of large-scale, multiplexed barcode sequencing experiments. The procedure outlined here was used to score interactions for ~4 million chemical-by-mutant combinations in our recently published chemical-genetic interaction screen of nearly 14,000 chemical compounds across seven diverse compound collections. Here we selected a representative subset of these data on which to demonstrate our analysis pipeline. BEAN-counter is open source, written in Python, and freely available for academic use. Users should be proficient at the command line; advanced users who wish to analyze larger datasets with hundreds or more conditions should also be familiar with concepts in analysis of high-throughput biological data. BEAN-counter encapsulates the knowledge we have accumulated from, and successfully applied to, our multiplexed, pooled barcode sequencing experiments. This protocol will be useful to those interested in generating their own high-dimensional, quantitative characterizations of gene or chemical function in a high-throughput manner.

PMID: 30635653 [PubMed - indexed for MEDLINE]



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Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species-dependent essential genes.

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Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species-dependent essential genes.

Genome Res. 2019 Jan 11;:

Authors: Sanchez MR, Payen C, Cheong F, Hovde BT, Bissonnette S, Arkin AP, Skerker JM, Brem RB, Caudy AA, Dunham MJ

Abstract
To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of S. cerevisiae We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with S. cerevisiae predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that trans-acting factors that are background-dependent contribute to differential gene essentiality. This study demonstrates that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotation. Our method can be widely applied in microorganisms to further our understanding of genome evolution.

PMID: 30635343 [PubMed - as supplied by publisher]



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