Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

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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Exploring targets of TET2-mediated methylation reprogramming as potential discriminators of prostate cancer progression.

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Exploring targets of TET2-mediated methylation reprogramming as potential discriminators of prostate cancer progression.

Clin Epigenetics. 2019 Mar 27;11(1):54

Authors: Kamdar S, Isserlin R, Van der Kwast T, Zlotta AR, Bader GD, Fleshner NE, Bapat B

Abstract
BACKGROUND: Global DNA methylation alterations are hallmarks of cancer. The tumor-suppressive TET enzymes, which are involved in DNA demethylation, are decreased in prostate cancer (PCa); in particular, TET2 is specifically targeted by androgen-dependent mechanisms of repression in PCa and may play a central role in carcinogenesis. Thus, the identification of key genes targeted by TET2 dysregulation may provide further insight into cancer biology.
RESULTS: Using a CRISPR/Cas9-derived TET2-knockout prostate cell line, and through whole-transcriptome and whole-methylome sequencing, we identified seven candidate genes-ASB2, ETNK2, MEIS2, NRG1, NTN1, NUDT10, and SRPX-exhibiting reduced expression and increased promoter methylation, a pattern characteristic of tumor suppressors. Decreased expression of these genes significantly discriminates between recurrent and non-recurrent prostate tumors from the Cancer Genome Atlas (TCGA) cohort (n = 423), and ASB2, NUDT10, and SRPX were significantly correlated with lower recurrence-free survival in patients by Kaplan-Meier analysis. ASB2, MEIS2, and SRPX also showed significantly lower expression in high-risk Gleason score 8 tumors as compared to low or intermediate risk tumors, suggesting that these genes may be particularly useful as indicators of PCa progression. Furthermore, methylation array probes in the TCGA dataset, which were proximal to the highly conserved, differentially methylated sites identified in our TET2-knockout cells, were able to significantly distinguish between matched prostate tumor and normal prostate tissues (n = 50 pairs). Except ASB2, all genes exhibited significantly increased methylation at these probes, and methylation status of at least one probe for each of these genes showed association with measures of PCa progression such as recurrence, stage, or Gleason score. Since ASB2 did not have any probes within the TET2-knockout differentially methylated region, we validated ASB2 methylation in an independent series of matched tumor-normal samples (n = 19) by methylation-specific qPCR, which revealed concordant and significant increases in promoter methylation within the TET2-knockout site.
CONCLUSIONS: Our study identifies seven genes governed by TET2 loss in PCa which exhibit an association between their methylation and expression status and measures of PCa progression. As differential methylation profiles and TET2 expression are associated with advanced PCa, further investigation of these specialized TET2 targets may provide important insights into patterns of carcinogenic gene dysregulation.

PMID: 30917865 [PubMed - in process]



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Automated, predictive, and interpretable inference of Caenorhabditis elegans escape dynamics.

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Automated, predictive, and interpretable inference of Caenorhabditis elegans escape dynamics.

Proc Natl Acad Sci U S A. 2019 04 09;116(15):7226-7231

Authors: Daniels BC, Ryu WS, Nemenman I

Abstract
The roundworm Caenorhabditis elegans exhibits robust escape behavior in response to rapidly rising temperature. The behavior lasts for a few seconds, shows history dependence, involves both sensory and motor systems, and is too complicated to model mechanistically using currently available knowledge. Instead we model the process phenomenologically, and we use the Sir Isaac dynamical inference platform to infer the model in a fully automated fashion directly from experimental data. The inferred model requires incorporation of an unobserved dynamical variable and is biologically interpretable. The model makes accurate predictions about the dynamics of the worm behavior, and it can be used to characterize the functional logic of the dynamical system underlying the escape response. This work illustrates the power of modern artificial intelligence to aid in discovery of accurate and interpretable models of complex natural systems.

PMID: 30902894 [PubMed - indexed for MEDLINE]



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Global Genetic Networks and the Genotype-to-Phenotype Relationship.

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Global Genetic Networks and the Genotype-to-Phenotype Relationship.

Cell. 2019 Mar 21;177(1):85-100

Authors: Costanzo M, Kuzmin E, van Leeuwen J, Mair B, Moffat J, Boone C, Andrews B

Abstract
Genetic interactions identify combinations of genetic variants that impinge on phenotype. With whole-genome sequence information available for thousands of individuals within a species, a major outstanding issue concerns the interpretation of allelic combinations of genes underlying inherited traits. In this Review, we discuss how large-scale analyses in model systems have illuminated the general principles and phenotypic impact of genetic interactions. We focus on studies in budding yeast, including the mapping of a global genetic network. We emphasize how information gained from work in yeast translates to other systems, and how a global genetic network not only annotates gene function but also provides new insights into the genotype-to-phenotype relationship.

PMID: 30901552 [PubMed - in process]



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