Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Bmi1 regulates human glioblastoma stem cells through activation of differential gene networks in CD133+ brain tumor initiating cells.

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Bmi1 regulates human glioblastoma stem cells through activation of differential gene networks in CD133+ brain tumor initiating cells.

J Neurooncol. 2019 May 21;:

Authors: Vora P, Seyfrid M, Venugopal C, Qazi MA, Salim S, Isserlin R, Subapanditha M, O'Farrell E, Mahendram S, Singh M, Bakhshinyan D, Chokshi C, McFarlane N, Dvorkin-Gheva A, Brown KR, Murty N, Moffat J, Bader GD, Singh SK

Abstract
PURPOSE: Glioblastoma (GBM) is the most aggressive adult brain cancer, with a 15 month median survivorship attributed to the existence of treatment-refractory brain tumor initiating cells (BTICs). In order to better understand the mechanisms regulating the tumorigenic properties of this population, we studied the role of the polycomb group member BMI1 in our patient-derived GBM BTICs and its relationship with CD133, a well-established marker of BTICs.
METHODS: Using gain and loss-of-function studies for Bmi1 in neural stem cells (NSCs) and patient-derived GBM BTICs respectively, we assessed in vitro self-renewal and in vivo tumor formation in these two cell populations. We further explored the BMI1 transcriptional regulatory network through RNA sequencing of different GBM BTIC populations that were knocked down for Bmi1.
RESULTS: There is a differential role of BMI1 in CD133-positive cells, notably involving cell metabolism. In addition, we identified pivotal targets downstream of BMI1 in CD133+ cells such as integrin alpha 2 (ITGA2), that may contribute to regulating GBM stem cell properties.
CONCLUSIONS: Our work sheds light on the association of three genes with CD133-BMI1 circuitry, their importance as downstream effectors of the BMI1 signalling pathway, and their potential as future targets for tackling GBM treatment-resistant cell populations.

PMID: 31115870 [PubMed - as supplied by publisher]



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The Long Noncoding RNA Pnky Is a Trans-acting Regulator of Cortical Development In Vivo.

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The Long Noncoding RNA Pnky Is a Trans-acting Regulator of Cortical Development In Vivo.

Dev Cell. 2019 May 20;49(4):632-642.e7

Authors: Andersen RE, Hong SJ, Lim JJ, Cui M, Harpur BA, Hwang E, Delgado RN, Ramos AD, Liu SJ, Blencowe BJ, Lim DA

Abstract
While it is now appreciated that certain long noncoding RNAs (lncRNAs) have important functions in cell biology, relatively few have been shown to regulate development in vivo, particularly with genetic strategies that establish cis versus trans mechanisms. Pnky is a nuclear-enriched lncRNA that is transcribed divergently from the neighboring proneural transcription factor Pou3f2. Here, we show that conditional deletion of Pnky from the developing cortex regulates the production of projection neurons from neural stem cells (NSCs) in a cell-autonomous manner, altering postnatal cortical lamination. Surprisingly, Pou3f2 expression is not disrupted by deletion of the entire Pnky gene. Moreover, expression of Pnky from a BAC transgene rescues the differential gene expression and increased neurogenesis of Pnky-knockout NSCs, as well as the developmental phenotypes of Pnky-deletion in vivo. Thus, despite being transcribed divergently from a key developmental transcription factor, the lncRNA Pnky regulates development in trans.

PMID: 31112699 [PubMed - in process]



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Characterizing ABC-Transporter Substrate-Likeness Using a Clean-Slate Genetic Background.

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Characterizing ABC-Transporter Substrate-Likeness Using a Clean-Slate Genetic Background.

Front Pharmacol. 2019;10:448

Authors: Sokolov A, Ashenden S, Sahin N, Lewis R, Erdem N, Ozaltan E, Bender A, Roth FP, Cokol M

Abstract
Mutations in ATP Binding Cassette (ABC)-transporter genes can have major effects on the bioavailability and toxicity of the drugs that are ABC-transporter substrates. Consequently, methods to predict if a drug is an ABC-transporter substrate are useful for drug development. Such methods traditionally relied on literature curated collections of ABC-transporter dependent membrane transfer assays. Here, we used a single large-scale dataset of 376 drugs with relative efficacy on an engineered yeast strain with all ABC-transporter genes deleted (ABC-16), to explore the relationship between a drug's chemical structure and ABC-transporter substrate-likeness. We represented a drug's chemical structure by an array of substructure keys and explored several machine learning methods to predict the drug's efficacy in an ABC-16 yeast strain. Gradient-Boosted Random Forest models outperformed all other methods with an AUC of 0.723. We prospectively validated the model using new experimental data and found significant agreement with predictions. Our analysis expands the previously reported chemical substructures associated with ABC-transporter substrates and provides an alternative means to investigate ABC-transporter substrate-likeness.

PMID: 31105571 [PubMed]



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A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction.

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A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction.

Elife. 2019 May 14;8:

Authors: Afshar Bakooshli M, Lippmann ES, Mulcahy B, Iyer N, Nguyen CT, Tung K, Stewart BA, van den Dorpel H, Fuehrmann T, Shoichet M, Bigot A, Pegoraro E, Ahn H, Ginsberg H, Zhen M, Ashton RS, Gilbert PM

Abstract
Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.

PMID: 31084710 [PubMed - in process]



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Effect of Sugar 2',4'-Modifications on Gene Silencing Activity of Small Interfering RNA Duplexes.

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Effect of Sugar 2',4'-Modifications on Gene Silencing Activity of Small Interfering RNA Duplexes.

Nucleic Acid Ther. 2019 May 14;:

Authors: Malek-Adamian E, Fakhoury J, Arnold AE, Martínez-Montero S, Shoichet MS, Damha MJ

Abstract
In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2',4') modification was assessed by comparing gene silencing ability to 2'- or 4'- (singly) modified siRNA counterparts. siRNAs with (2',4')-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2'- or 4'-modified overhang, suggesting that 2',4'-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2'-F,4'-OMe-ribose or 2'-F,4'-OMe-arabinose at the overhangs. When modifications were placed at both overhangs and internal positions, a duplex with the 2'-F (internal) and 2'-F,4'-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2'-OMe (internal) and 2',4'-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2',4'-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.

PMID: 31084536 [PubMed - as supplied by publisher]



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Cationic block amphiphiles show anti-mitochondrial activity in multi-drug resistant breast cancer cells.

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Cationic block amphiphiles show anti-mitochondrial activity in multi-drug resistant breast cancer cells.

J Control Release. 2019 May 06;:

Authors: Czupiel PP, Delplace V, Shoichet MS

Abstract
Currently, there are limited treatment options for multi-drug resistant breast cancer. Lipid modified, cationic peptides have the potential to reach the mitochondria, which are attractive targets for the treatment of multi-drug resistant (MDR) breast cancer; yet, little is known about their mitochondrial targeting and anti-cancer activity. Interestingly, lipid modified cationic peptides, typically used as gene transfection agents, exhibit similar structural features to mitochondrial targeted peptides. Using octahistidine-octaarginine (H8R8) as a model cationic peptide for cell penetration and endosomal escape, we explored the anti-cancer potential of lipid-modified cationic peptides as a function of amphiphilicity, biodegradability and lipid structure. We found that cationic peptides modified with a lipid that is at least 12 carbons in length exhibit potent anti-cancer activity in the low micromolar range in both EMT6/P and EMT6/AR-1 breast cancer cells. Comparing degradable and non-degradable linkers, as well as L- and D-amino acid sequences, we found that the anti-cancer activity is mostly independent of the biodegradation of the lipid modified cationic peptides. Two candidates, stearyl-H8R8 (Str-H8R8) and vitamin E succinate-H8R8 (VES-H8R8) were cytotoxic to cancer cells by mitochondria depolarization. We observed increased reactive oxygen species (ROS) production, reduced cell bioenergetics and drug efflux, triggering apoptosis and G1 cell cycle arrest. Compared to Str-H8R8, VES-H8R8 showed enhanced cancer cell selectivity and drug efflux inhibition, thereby serving as a potential novel therapeutic agent. This study deepens our understanding of lipid modified cationic peptides and uncovers their potential in multi-drug resistant breast cancer.

PMID: 31071370 [PubMed - as supplied by publisher]



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Non-steric-zipper models for pathogenic α-synuclein conformers.

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Non-steric-zipper models for pathogenic α-synuclein conformers.

APL Bioeng. 2018 Jun;2(2):026105

Authors: Schuman B, Won A, Brand-Arzamendi K, Koprich JB, Wen XY, Howson PA, Brotchie JM, Yip CM

Abstract
Parkinson's disease neurodegenerative brain tissue exhibits two biophysically distinct α-synuclein fiber isoforms-single stranded fibers that appear to be steric-zippers and double-stranded fibers with an undetermined structure. Herein, we describe a β-helical homology model of α-synuclein that exhibits stability in probabilistic and Monte Carlo simulations as a candidate for stable prional dimer conformers in equilibrium with double-stranded fibers and cytotoxic pore assemblies. Molecular models of β-helical pore assemblies are consistent with α-synucleinA53T transfected rat immunofluorescence epitope maps. Atomic force microscopy reveals that α-synuclein peptides aggregate into anisotropic fibrils lacking the density or circumference of a steric-zipper. Moreover, fibrillation was blocked by mutations designed to hinder β-helical but not steric-zipper conformations. β-helical species provide a structural basis for previously described biophysical properties that are incompatible with a steric-zipper, provide pathogenic mechanisms for familial human α-synuclein mutations, and offer a direct cytotoxic target for therapeutic development.

PMID: 31069302 [PubMed]



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Correction to: A microfluidic platform for continuous monitoring of dopamine homeostasis in dopaminergic cells.

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Correction to: A microfluidic platform for continuous monitoring of dopamine homeostasis in dopaminergic cells.

Microsyst Nanoeng. 2019;5:19

Authors: Yu Y, de Campos RPS, Hong S, Krastev DL, Sadanand S, Leung Y, Wheeler AR

Abstract
[This corrects the article DOI: 10.1038/s41378-019-0049-2.].

PMID: 31063161 [PubMed]



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A microfluidic platform for continuous monitoring of dopamine homeostasis in dopaminergic cells.

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A microfluidic platform for continuous monitoring of dopamine homeostasis in dopaminergic cells.

Microsyst Nanoeng. 2019;5:10

Authors: Yu Y, de Campos RPS, Hong S, Krastev DL, Sadanand S, Leung Y, Wheeler AR

Abstract
Homeostasis of dopamine, a classical neurotransmitter, is a key indicator of neuronal health. Dysfunction in the regulation of dopamine is implicated in a long list of neurological disorders, including addiction, depression, and neurodegeneration. The existing methods used to evaluate dopamine homeostasis in vitro are inconvenient and do not allow for continuous non-destructive measurement. In response to this challenge, we introduce an integrated microfluidic system that combines dopaminergic cell culture and differentiation with electroanalytical measurements of extracellular dopamine in real-time at any point during an assay. We used the system to examine the behavior of differentiated SH-SY5Y cells upon exposure to four dopamine transporter ant/agonists (cocaine, ketamine, epigallocatechin gallate, and amphetamine) and study their pharmacokinetics. The IC50 values of cocaine, ketamine, and epigallocatechin gallate were determined to be (average ± standard deviation) 3.7 ± 1.1 µM, 51.4 ± 17.9 µM, and 2.6 ± 0.8 µM, respectively. Furthermore, we used the new system to study amphetamine-mediated dopamine release to probe the related phenomena of dopamine transporter-mediated reverse-transport and dopamine release from vesicles. We propose that this platform, which is the first platform to simultaneously evaluate uptake and release, could be useful to screen for drugs and other agents that target dopaminergic neurons and the function of the dopamine transporter. More broadly, this platform should be adaptable for any application that could benefit from high-temporal resolution electroanalysis combined with multi-day cell culture using small numbers of cells.

PMID: 31057937 [PubMed]



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Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate the Infarcted Pig Heart but Induce Ventricular Tachyarrhythmias.

Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate the Infarcted Pig Heart but Induce Ventricular Tachyarrhythmias.

Stem Cell Reports. 2019 Apr 22;:

Authors: Romagnuolo R, Masoudpour H, Porta-Sánchez A, Qiang B, Barry J, Laskary A, Qi X, Massé S, Magtibay K, Kawajiri H, Wu J, Valdman Sadikov T, Rothberg J, Panchalingam KM, Titus E, Li RK, Zandstra PW, Wright GA, Nanthakumar K, Ghugre NR, Keller G, Laflamme MA

Abstract
Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show considerable promise for regenerating injured hearts, and we therefore tested their capacity to stably engraft in a translationally relevant preclinical model, the infarcted pig heart. Transplantation of immature hESC-CMs resulted in substantial myocardial implants within the infarct scar that matured over time, formed vascular networks with the host, and evoked minimal cellular rejection. While arrhythmias were rare in infarcted pigs receiving vehicle alone, hESC-CM recipients experienced frequent monomorphic ventricular tachycardia before reverting back to normal sinus rhythm by 4 weeks post transplantation. Electroanatomical mapping and pacing studies implicated focal mechanisms, rather than macro-reentry, for these graft-related tachyarrhythmias as evidenced by an abnormal centrifugal pattern with earliest electrical activation in histologically confirmed graft tissue. These findings demonstrate the suitability of the pig model for the preclinical development of a hESC-based cardiac therapy and provide new insights into the mechanistic basis of electrical instability following hESC-CM transplantation.

PMID: 31056479 [PubMed - as supplied by publisher]



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