Donnelly Centre for Cellular and Biomolecular Research

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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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Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

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Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

Mol Cell. 2019 Apr 26;:

Authors: Ali I, Ruiz DG, Ni Z, Johnson JR, Zhang H, Li PC, Khalid MM, Conrad RJ, Guo X, Min J, Greenblatt J, Jacobson M, Krogan NJ, Ott M

Abstract
Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

PMID: 31054975 [PubMed - as supplied by publisher]



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A Unified Resource for Tracking Anti-CRISPR Names.

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A Unified Resource for Tracking Anti-CRISPR Names.

CRISPR J. 2018 Oct;1:304-305

Authors: Bondy-Denomy J, Davidson AR, Doudna JA, Fineran PC, Maxwell KL, Moineau S, Peng X, Sontheimer EJ, Wiedenheft B

PMID: 31021273 [PubMed - in process]



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Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

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Meet the Anti-CRISPRs: Widespread Protein Inhibitors of CRISPR-Cas Systems.

CRISPR J. 2019 Feb;2:23-30

Authors: Hwang S, Maxwell KL

Abstract
The constant selective pressure exerted by phages, the viruses that infect bacteria, has led to the evolution of a wide range of anti-phage defenses. One of these defense mechanisms, CRISPR-Cas, provides an adaptive immune system to battle phage infection and inhibit horizontal gene transfer by plasmids, transposons, and other mobile genetic elements. Although CRISPR-Cas systems are widespread in bacteria and archaea, they appear to have minimal long-term evolutionary effects with respect to limiting horizontal gene transfer. One factor that may contribute to this may be the presence of potent inhibitors of CRISPR-Cas systems, known as anti-CRISPR proteins. Forty unique families of anti-CRISPR proteins have been described to date. These inhibitors, which are active against both Class 1 and 2 CRISPR-Cas systems, have a wide range of mechanisms of activity. Studies of these proteins have provided important insight into the evolutionary arms race between bacteria and phages, and have contributed to the development of biotechnological tools that can be harnessed for control of CRISPR-Cas genome editing.

PMID: 31021234 [PubMed - in process]



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scClustViz - Single-cell RNAseq cluster assessment and visualization.

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scClustViz - Single-cell RNAseq cluster assessment and visualization.

F1000Res. 2018;7:

Authors: Innes BT, Bader GD

Abstract
Single-cell RNA sequencing (scRNAseq) represents a new kind of microscope that can measure the transcriptome profiles of thousands of individual cells from complex cellular mixtures, such as in a tissue, in a single experiment. This technology is particularly valuable for characterization of tissue heterogeneity because it can be used to identify and classify all cell types in a tissue. This is generally done by clustering the data, based on the assumption that cells of a particular type share similar transcriptomes, distinct from other cell types in the tissue. However, nearly all clustering algorithms have tunable parameters which affect the number of clusters they will identify in data. The R Shiny software tool described here, scClustViz, provides a simple interactive graphical user interface for exploring scRNAseq data and assessing the biological relevance of clustering results. Given that cell types are expected to have distinct gene expression patterns, scClustViz uses differential gene expression between clusters as a metric for assessing the fit of a clustering result to the data at multiple cluster resolution levels. This helps select a clustering parameter for further analysis. scClustViz also provides interactive visualisation of: cluster-specific distributions of technical factors, such as predicted cell cycle stage and other metadata; cluster-wise gene expression statistics to simplify annotation of cell types and identification of cell type specific marker genes; and gene expression distributions over all cells and cell types. scClustViz provides an interactive interface for visualisation, assessment, and biological interpretation of cell-type classifications in scRNAseq experiments that can be easily added to existing analysis pipelines, enabling customization by bioinformaticians while enabling biologists to explore their results without the need for computational expertise. It is available at https://baderlab.github.io/scClustViz/.

PMID: 31016009 [PubMed - in process]



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