Donnelly Centre for Cellular and Biomolecular Research

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Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models.

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Blockade of TGF-β signaling with novel synthetic antibodies limits immune exclusion and improves chemotherapy response in metastatic ovarian cancer models.

Oncoimmunology. 2019;8(2):e1539613

Authors: Newsted D, Banerjee S, Watt K, Nersesian S, Truesdell P, Blazer LL, Cardarelli L, Adams JJ, Sidhu SS, Craig AW

Abstract
Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. EOC is often diagnosed at late stages, with peritoneal metastases and ascites production. Current surgery and platinum-based chemotherapy regimes fail to prevent recurrence in most patients. High levels of Transforming growth factor-β (TGF-β) within ascites has been linked to poor prognosis. TGF-β signaling promotes epithelial-mesenchymal transition (EMT) in EOC tumor cells, and immune suppression within the tumor microenvironment, with both contributing to chemotherapy resistance and metastasis. The goal of this study was to develop specific synthetic inhibitory antibodies to the Type II TGF-β receptor (TGFBR2), and test these antibodies in EOC cell and tumor models. Following screening of a phage-displayed synthetic antigen-binding fragment (Fab) library with the extracellular domain of TGFBR2, we identified a lead inhibitory Fab that suppressed TGF-β signaling in mouse and human EOC cell lines. Affinity maturation of the lead inhibitory Fab resulted in several derivative Fabs with increased affinity for TGFBR2 and efficacy as suppressors of TGF-β signaling, EMT and EOC cell invasion. In EOC xenograft and syngeneic tumor models, blockade of TGFBR2 with our lead antibodies led to improved chemotherapy response. This correlated with reversal of EMT and immune exclusion in these tumor models with TGFBR2 blockade. Together, these results describe new inhibitors of the TGF-β pathway that improve antitumor immunity, and response to chemotherapy in preclinical EOC models.

PMID: 30713798 [PubMed]



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Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

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Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

Structure. 2019 Jan 17;:

Authors: Teyra J, Singer AU, Schmitges FW, Jaynes P, Kit Leng Lui S, Polyak MJ, Fodil N, Krieger JR, Tong J, Schwerdtfeger C, Brasher BB, Ceccarelli DFJ, Moffat J, Sicheri F, Moran MF, Gros P, Eichhorn PJA, Lenter M, Boehmelt G, Sidhu SS

Abstract
The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor β pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

PMID: 30713027 [PubMed - as supplied by publisher]



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Ideation and implementation of an open science drug discovery business model - M4K Pharma.

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Ideation and implementation of an open science drug discovery business model - M4K Pharma.

Wellcome Open Res. 2018;3:154

Authors: Morgan MR, Roberts OG, Edwards AM

Abstract
M4K Pharma was incorporated to launch an open science drug discovery program that relies on regulatory exclusivity as its primary intellectual property and commercial asset, in lieu of patents.In many cases and in key markets, using regulatory exclusivity can provide equivalent commercial protection to patents, while also being compatible with open science. The model is proving attractive to government, foundation and individual funders, who collectively have different expectations for returns on investment compared with biotech, pharmaceutical companies, or venture capital investors.In the absence of these investor-driven requirements for returns, it should be possible to commercialize therapeutics at affordable prices.M4K is piloting this open science business model in a rare paediatric brain tumour, but there is no reason it should not be more widely applicable.

PMID: 30705971 [PubMed]



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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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