Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

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A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

EMBO J. 2019 Apr 15;:

Authors: Maslon MM, Braunschweig U, Aitken S, Mann AR, Kilanowski F, Hunter CJ, Blencowe BJ, Kornblihtt AR, Adams IR, Cáceres JF

Abstract
The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

PMID: 30988016 [PubMed - as supplied by publisher]



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Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector.

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Establishment of an erythroid progenitor cell line capable of enucleation achieved with an inducible c-Myc vector.

BMC Biotechnol. 2019 Apr 15;19(1):21

Authors: Mayers S, Moço PD, Maqbool T, Silva PN, Kilkenny DM, Audet J

Abstract
BACKGROUND: A robust scalable method for producing enucleated red blood cells (RBCs) is not only a process to produce packed RBC units for transfusion but a potential platform to produce modified RBCs with applications in advanced cellular therapy. Current strategies for producing RBCs have shortcomings in the limited self-renewal capacity of progenitor cells, or difficulties in effectively enucleating erythroid cell lines. We explored a new method to produce RBCs by inducibly expressing c-Myc in primary erythroid progenitor cells and evaluated the proliferative and maturation potential of these modified cells.
RESULTS: Primary erythroid progenitor cells were genetically modified with an inducible gene transfer vector expressing a single transcription factor, c-Myc, and all the gene elements required to achieve dox-inducible expression. Genetically modified cells had enhanced proliferative potential compared to control cells, resulting in exponential growth for at least 6 weeks. Inducibly proliferating erythroid (IPE) cells were isolated with surface receptors similar to colony forming unit-erythroid (CFU-Es), and after removal of ectopic c-Myc expression cells hemoglobinized, decreased in cell size to that of native RBCs, and enucleated achieving cultures with 17% enucleated cells. Experiments with IPE cells at various levels of ectopic c-Myc expression provided insight into differentiation dynamics of the modified cells, and an optimized two-stage differentiation strategy was shown to promote greater expansion and maturation.
CONCLUSIONS: Genetic engineering of adult erythroid progenitor cells with an inducible c-Myc vector established an erythroid progenitor cell line that could produce RBCs, demonstrating the potential of this approach to produce large quantities of RBCs and modified RBC products.

PMID: 30987611 [PubMed - in process]



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Genetic interaction networks in cancer cells.

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Genetic interaction networks in cancer cells.

Curr Opin Genet Dev. 2019 Apr 08;54:64-72

Authors: Mair B, Moffat J, Boone C, Andrews BJ

Abstract
The genotype-to-phenotype relationship in health and disease is complex and influenced by both an individual's environment and their unique genome. Personal genetic variants can modulate gene function to generate a phenotype either through a single gene effect or through genetic interactions involving two or more genes. The relevance of genetic interactions to disease phenotypes has been particularly clear in cancer research, where an extreme genetic interaction, synthetic lethality, has been exploited as a therapeutic strategy. The obvious benefits of unmasking genetic background-specific vulnerabilities, coupled with the power of systematic genome editing, have fueled efforts to translate genetic interaction mapping from model organisms to human cells. Here, we review recent developments in genetic interaction mapping, with a focus on CRISPR-based genome editing technologies and cancer.

PMID: 30974317 [PubMed - as supplied by publisher]



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Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies.

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Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies.

Cell Rep. 2019 Apr 09;27(2):599-615.e12

Authors: Mair B, Tomic J, Masud SN, Tonge P, Weiss A, Usaj M, Tong AHY, Kwan JJ, Brown KR, Titus E, Atkins M, Chan KSK, Munsie L, Habsid A, Han H, Kennedy M, Cohen B, Keller G, Moffat J

Abstract
Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.

PMID: 30970261 [PubMed - in process]



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Exploiting DNA Replication Stress for Cancer Treatment.

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Exploiting DNA Replication Stress for Cancer Treatment.

Cancer Res. 2019 Apr 09;:

Authors: Ubhi T, Brown GW

Abstract
Complete and accurate DNA replication is fundamental to cellular proliferation and genome stability. Obstacles that delay, prevent, or terminate DNA replication cause the phenomena termed DNA replication stress. Cancer cells exhibit chronic replication stress due to the loss of proteins that protect or repair stressed replication forks and due to the continuous proliferative signaling, providing an exploitable therapeutic vulnerability in tumors. Here, we outline current and pending therapeutic approaches leveraging tumor-specific replication stress as a target, in addition to the challenges associated with such therapies. We discuss how replication stress modulates the cell-intrinsic innate immune response and highlight the integration of replication stress with immunotherapies. Together, exploiting replication stress for cancer treatment seems to be a promising strategy as it provides a selective means of eliminating tumors, and with continuous advances in our knowledge of the replication stress response and lessons learned from current therapies in use, we are moving toward honing the potential of targeting replication stress in the clinic.

PMID: 30967400 [PubMed - as supplied by publisher]



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Velocity Saturation in Digital Microfluidics.

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Velocity Saturation in Digital Microfluidics.

Langmuir. 2019 Apr 08;:

Authors: Swyer I, Fobel R, Wheeler AR

Abstract
In digital microfluidics, discrete droplets of fluid are made to move on an open surface with no microchannels. These systems are commonly operated by application of electrical driving forces to an array of electrodes. While these driving forces are well characterized, the dissipative forces opposing droplet movement have not been as thoroughly examined. In recognition of this deficit, we used force-velocity plots to characterize droplet movement in digital microfluidics, which was found to be consistent with a simple theoretical framework for understanding dissipation effects for droplets in two-plate, air-filled devices. Interestingly, in some conditions, a previously unreported ″velocity saturation″ effect was observed. When examined across a range of different liquids, the forces at which this saturation occurs seem to be lower for liquids with smaller surface tensions. Furthermore, when driven at forces that cause saturation, physical phenomena are observed that are akin to what has been reported for stationary droplets in the electrowetting literature. These phenomena are detrimental to device performance, leading to a new "force window" approach that delineates the optimum operation conditions for different liquids. We propose that these findings may be useful for a wide range of applications for experts and new users alike in this growing field.

PMID: 30958677 [PubMed - as supplied by publisher]



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Testing cancer inhibitors at scale.

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Testing cancer inhibitors at scale.

Nat Biomed Eng. 2018 Apr;2(4):203-204

Authors: Saraon P, Grozavu I, Stagljar I

PMID: 30936440 [PubMed - in process]



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The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids.

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The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids.

Cell Stem Cell. 2019 Mar 19;:

Authors: Seneviratne AK, Xu M, Henao JJA, Fajardo VA, Hao Z, Voisin V, Xu GW, Hurren R, Kim S, MacLean N, Wang X, Gronda M, Jeyaraju D, Jitkova Y, Ketela T, Mullokandov M, Sharon D, Thomas G, Chouinard-Watkins R, Hawley JR, Schafer C, Yau HL, Khuchua Z, Aman A, Al-Awar R, Gross A, Claypool SM, Bazinet R, Lupien M, Chan S, De Carvalho DD, Minden MD, Bader GD, Stark KD, LeBlanc P, Schimmer AD

Abstract
Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

PMID: 30930145 [PubMed - as supplied by publisher]



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Potent Neutralization of Staphylococcal Enterotoxin B in vivo by Antibodies that Block Binding to the T-Cell Receptor.

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Potent Neutralization of Staphylococcal Enterotoxin B in vivo by Antibodies that Block Binding to the T-Cell Receptor.

J Mol Biol. 2019 Mar 27;:

Authors: Chen G, Karauzum H, Long H, Carranza D, Holtsberg FW, Howell KA, Abaandou L, Zhang B, Jarvik N, Ye W, Liao GC, Gross ML, Leung DW, Amarasinghe GK, Aman MJ, Sidhu SS

Abstract
To develop an antibody (Ab) therapeutic against Staphylococcal Enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a "Class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed "Class M" Abs. A systematic engineering approach was applied to affinity mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) revealed that the Class T epitope on SEB overlapped with the T-cell receptor (TCR) binding site, whereas other evidence suggested that the Class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ~50-fold more potent toxin-neutralizing efficacy than the best Class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two Class M binding sites and two Class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that Class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bispecific Ab engineering.

PMID: 30928493 [PubMed - as supplied by publisher]



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15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection.

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15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection.

PLoS Pathog. 2019 03;15(3):e1007597

Authors: Evans RJ, Pline K, Loynes CA, Needs S, Aldrovandi M, Tiefenbach J, Bielska E, Rubino RE, Nicol CJ, May RC, Krause HM, O'Donnell VB, Renshaw SA, Johnston SA

Abstract
Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.

PMID: 30921435 [PubMed - indexed for MEDLINE]



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