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Recent Publications

Mechanisms of suppression: The wiring of genetic resilience.

Mechanisms of suppression: The wiring of genetic resilience.

Bioessays. 2017 Jun 05;:

Authors: van Leeuwen J, Pons C, Boone C, Andrews BJ

Abstract
Recent analysis of genome sequences has identified individuals that are healthy despite carrying severe disease-associated mutations. A possible explanation is that these individuals carry a second genomic perturbation that can compensate for the detrimental effects of the disease allele, a phenomenon referred to as suppression. In model organisms, suppression interactions are generally divided into two classes: genomic suppressors which are secondary mutations in the genome that bypass a mutant phenotype, and dosage suppression interactions in which overexpression of a suppressor gene rescues a mutant phenotype. Here, we describe the general properties of genomic and dosage suppression, with an emphasis on the budding yeast. We propose that suppression interactions between genetic variants are likely relevant for determining the penetrance of human traits. Consequently, an understanding of suppression mechanisms may guide the discovery of protective variants in healthy individuals that carry disease alleles, which could direct the rational design of new therapeutics.

PMID: 28582599 [PubMed - as supplied by publisher]



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Engineering cell fitness: lessons for regenerative medicine.

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Engineering cell fitness: lessons for regenerative medicine.

Curr Opin Biotechnol. 2017 May 25;47:7-15

Authors: Shakiba N, Zandstra PW

Abstract
Cell competition results in the loss of weaker cells and the dominance of stronger cells. So-called 'loser' cells are either removed by active elimination or by limiting their access to survival factors. Recently, competition has been shown to serve as a surveillance mechanism against emerging aberrant cells in both the developing and adult organism, contributing to overall organism fitness and survival. Here, we explore the origins and implications of cell competition in development, tissue homeostasis, and in vitro culture. We also provide a forward look on the use of cell competition to interpret multicellular dynamics while offering a perspective on harnessing competition to engineer cells with optimized and controllable fitness characteristics for regenerative medicine applications.

PMID: 28551499 [PubMed - as supplied by publisher]



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Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants.

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Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants.

PLoS Pathog. 2017 May 18;13(5):e1006372

Authors: Zhang W, Bailey-Elkin BA, Knaap RCM, Khare B, Dalebout TJ, Johnson GG, van Kasteren PB, McLeish NJ, Gu J, He W, Kikkert M, Mark BL, Sidhu SS

Abstract
The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques.

PMID: 28542609 [PubMed - as supplied by publisher]



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Identifying pathogenicity of human variants via paralog-based yeast complementation.

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Identifying pathogenicity of human variants via paralog-based yeast complementation.

PLoS Genet. 2017 May 25;13(5):e1006779

Authors: Yang F, Sun S, Tan G, Costanzo M, Hill DE, Vidal M, Andrews BJ, Boone C, Roth FP

Abstract
To better understand the health implications of personal genomes, we now face a largely unmet challenge to identify functional variants within disease-associated genes. Functional variants can be identified by trans-species complementation, e.g., by failure to rescue a yeast strain bearing a mutation in an orthologous human gene. Although orthologous complementation assays are powerful predictors of pathogenic variation, they are available for only a few percent of human disease genes. Here we systematically examine the question of whether complementation assays based on paralogy relationships can expand the number of human disease genes with functional variant detection assays. We tested over 1,000 paralogous human-yeast gene pairs for complementation, yielding 34 complementation relationships, of which 33 (97%) were novel. We found that paralog-based assays identified disease variants with success on par with that of orthology-based assays. Combining all homology-based assay results, we found that complementation can often identify pathogenic variants outside the homologous sequence region, presumably because of global effects on protein folding or stability. Within our search space, paralogy-based complementation more than doubled the number of human disease genes with a yeast-based complementation assay for disease variation.

PMID: 28542158 [PubMed - as supplied by publisher]



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Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence.

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Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence.

Nat Commun. 2017 May 25;8:15380

Authors: Rahman N, Brauer PM, Ho L, Usenko T, Tewary M, Zúñiga-Pflücker JC, Zandstra PW

Abstract
Efforts to recapitulate haematopoiesis, a process guided by spatial and temporal inductive signals, to generate haematopoietic progenitors from human pluripotent stem cells (hPSCs) have focused primarily on exogenous signalling pathway activation or inhibition. Here we show haemogenic niches can be engineered using microfabrication strategies by micropatterning hPSC-derived haemogenic endothelial (HE) cells into spatially-organized, size-controlled colonies. CD34+VECAD+ HE cells were generated with multi-lineage potential in serum-free conditions and cultured as size-specific haemogenic niches that displayed enhanced blood cell induction over non-micropatterned cultures. Intra-colony analysis revealed radial organization of CD34 and VECAD expression levels, with CD45+ blood cells emerging primarily from the colony centroid area. We identify the induced interferon gamma protein (IP-10)/p-38 MAPK signalling pathway as the mechanism for haematopoietic inhibition in our culture system. Our results highlight the role of spatial organization in hPSC-derived blood generation, and provide a quantitative platform for interrogating molecular pathways that regulate human haematopoiesis.

PMID: 28541275 [PubMed - in process]



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A novel immunotherapy targeting MMP-14 limits hypoxia, immune suppression and metastasis in triple-negative breast cancer models.

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A novel immunotherapy targeting MMP-14 limits hypoxia, immune suppression and metastasis in triple-negative breast cancer models.

Oncotarget. 2017 May 09;:

Authors: Ling B, Watt K, Banerjee S, Newsted D, Truesdell P, Adams J, Sidhu SS, Craig AW

Abstract
Matrix metalloproteinase-14 (MMP-14) is a clinically relevant target in metastatic cancers due to its role in tumor progression and metastasis. Since active MMP-14 is localized on the cell surface, it is amenable to antibody-mediated blockade in cancer, and here we describe our efforts to develop novel inhibitory anti-MMP-14 antibodies. A phage-displayed synthetic humanized Fab library was screened against the extracellular domain of MMP-14 and a panel of MMP14-specific Fabs were identified. A lead antibody that inhibits the catalytic domain of MMP-14 (Fab 3369) was identified and treatment of MDA-MB-231 breast cancer cells with Fab 3369 led to significant loss of extracellular matrix degradation and cell invasion abilities. In mammary orthotopic tumor xenograft assays, MMP-14 blockade by IgG 3369 limited tumor growth and metastasis. Analysis of tumor tissue sections revealed that MMP-14 blockade limited tumor neoangiogenesis and hypoxia. Similar effects of MMP-14 blockade in syngeneic 4T1 mammary tumors were observed, along with increased detection of cytotoxic immune cell markers. In conclusion, we show that immunotherapies targeting MMP-14 can limit immune suppression, tumor progression, and metastasis in triple-negative breast cancer.

PMID: 28537911 [PubMed - as supplied by publisher]



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Building a central nervous system: The neural stem cell lineage revealed.

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Building a central nervous system: The neural stem cell lineage revealed.

Neurogenesis (Austin). 2017;4(1):e1300037

Authors: Xu W, Lakshman N, Morshead CM

Abstract
Neural stem cells (NSCs) are a multipotent, self-renewing source of undifferentiated cells in the periventricular region of the mammalian central nervous system (CNS). Since their original discovery 25 years ago, much has been learned about their development, persistence, localization, properties and potential. Herein we discuss the current state of knowledge pertaining to neural stem cells with a focus on the lineage relationship between two NSC populations along the neuraxis and their regionally distinct niches in the CNS.

PMID: 28516107 [PubMed - in process]



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Deletion of α5 nicotine receptor subunits abolishes nicotinic aversive motivational effects in a manner that phenocopies dopamine receptor antagonism.

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Deletion of α5 nicotine receptor subunits abolishes nicotinic aversive motivational effects in a manner that phenocopies dopamine receptor antagonism.

Eur J Neurosci. 2017 May 12;:

Authors: Grieder TE, George O, Yee M, Bergamini MA, Chwalek M, Maal-Bared G, Vargas-Perez H, van der Kooy D

Abstract
Nicotine addiction is a worldwide epidemic that claims millions of lives each year. Genetic deletion of α5 nicotinic acetylcholine receptor (nAChR) subunits has been associated with increased nicotine intake, however, it remains unclear whether acute nicotine is less aversive or more rewarding, and whether mice lacking the α5 nAChR subunit can experience withdrawal from chronic nicotine. We used place conditioning and conditioned taste avoidance paradigms to examine the effect of α5 subunit-containing nAChR deletion (α5 -/-) on conditioned approach and avoidance behaviour in nondependent and nicotine-dependent and -withdrawn mice, and compared these motivational effects with those elicited after dopamine receptor antagonism. We show that nondependent α5 -/- mice find low, non-motivational doses of nicotine rewarding, and do not show an aversive conditioned response or taste avoidance to higher aversive doses of nicotine. Furthermore, nicotine-dependent α5 -/- mice do not show a conditioned aversive motivational response to withdrawal from chronic nicotine, although they continue to exhibit a somatic withdrawal syndrome. These effects phenocopy those observed after dopamine receptor antagonism, but are not additive, suggesting that α5 nAChR subunits act in the same pathway as dopamine and are critical for the experience of nicotine's aversive, but not rewarding motivational effects in both a nondependent and nicotine-dependent and -withdrawn motivational state. Genetic deletion of α5 nAChR subunits leads to a behavioural phenotype that exactly matches that observed after antagonizing dopamine receptors, thus we suggest that modulation of nicotinic receptors containing α5 subunits may modify dopaminergic signalling, suggesting novel therapeutic treatments for smoking cessation. This article is protected by copyright. All rights reserved.

PMID: 28498560 [PubMed - as supplied by publisher]



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P-Cadherin is necessary for retinal stem cell behavior in vitro, but not in vivo.

P-Cadherin is necessary for retinal stem cell behavior in vitro, but not in vivo.

Stem Cell Res. 2017 May 03;21:141-147

Authors: Coles BLK, van der Kooy D

Abstract
Adult retinal stem cells (RSCs) are rare quiescent cells within the ciliary epithelium of the eye, which is made up of non-pigmented N-Cadherin(+ve) inner and pigmented P-Cadherin(+ve) outer cell layers. Through FACs and single cell analyses, we have shown that RSCs arise from single cells from within the pigmented CE and express P-Cadherin. However, whether the expression of P-Cadherin is required for maintenance of the stem cell in vivo or in the formation of the clonal stem cell spheres in vitro is not known. Using cadherin functional blocking antibody experiments and a P-Cadherin (-/-) mouse to test whether the RSC population is affected by the loss of P-Cadherin expression, our experiments demonstrate that the RSCs reside in the pigmented CE layer and express P-Cadherin, which is important to the formation of adherent sphere colonies in vitro, however P-Cadherin is not required for maintenance of RSCs in vivo.

PMID: 28494434 [PubMed - as supplied by publisher]



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Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson Gilford Progeria Syndrome.

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Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson Gilford Progeria Syndrome.

Mol Cell Biol. 2017 May 08;:

Authors: Wheaton K, Campuzano D, Ma W, Sheinis M, Ho B, Brown GW, Benchimol S

Abstract
Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here, we show that young, cycling HGPS fibroblasts, exhibit chronic DNA damage primarily in S phase as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA altering its distribution away from replicating DNA in HGPS cells leading to γH2AX formation, ATR activation and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.

PMID: 28483909 [PubMed - as supplied by publisher]



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