Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

The Diverse Impacts of Phage Morons on Bacterial Fitness and Virulence.

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The Diverse Impacts of Phage Morons on Bacterial Fitness and Virulence.

Adv Virus Res. 2019;103:1-31

Authors: Taylor VL, Fitzpatrick AD, Islam Z, Maxwell KL

Abstract
The viruses that infect bacteria, known as phages, are the most abundant biological entity on earth. They play critical roles in controlling bacterial populations through phage-mediated killing, as well as through formation of bacterial lysogens. In this form, the survival of the phage depends on the survival of the bacterial host in which it resides. Thus, it is advantageous for phages to encode genes that contribute to bacterial fitness and expand the environmental niche. In many cases, these fitness factors also make the bacteria better able to survive in human infections and are thereby considered pathogenesis or virulence factors. The genes that encode these fitness factors, known as "morons," have been shown to increase bacterial fitness through a wide range of mechanisms and play important roles in bacterial diseases. This review outlines the benefits provided by phage morons in various aspects of bacterial life, including phage and antibiotic resistance, motility, adhesion and quorum sensing.

PMID: 30635074 [PubMed - indexed for MEDLINE]



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Phages Tune in to Host Cell Quorum Sensing.

Phages Tune in to Host Cell Quorum Sensing.

Cell. 2019 Jan 10;176(1-2):7-8

Authors: Maxwell KL

Abstract
Phages must be perfectly attuned to bacterial host cell physiology to ensure their optimal survival. Silpe and Bassler show that a Vibrio phage uses the host quorum-sensing pathway to trigger production of viral progeny at high cell density.

PMID: 30633910 [PubMed - in process]



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Local Delivery of BDNF Enables Behavioural Recovery and Tissue Repair in Stroke-Injured Rats.

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Local Delivery of BDNF Enables Behavioural Recovery and Tissue Repair in Stroke-Injured Rats.

Tissue Eng Part A. 2019 Jan 05;:

Authors: Obermeyer JM, Tuladhar A, Payne SL, Ho E, Morshead CM, Shoichet MS

Abstract
We induced tissue repair following ischemic stroke in rats by delivering brain-derived neurotropic factor (BDNF) directly to the brain. This protein is a potent modulator of plasticity and neuroprotection in the developing and adult central nervous system; however, the therapeutic potential of BDNF has been largely thwarted by its inability to cross the blood brain barrier at an effective concentration. Herein, we demonstrate enhanced acute recovery of forepaw dexterity and enhanced hindlimb function at 7 weeks post-injury by delivering BDNF locally, with sustained release in vivo for up to 21 days. Using an encapsulation-free methodology, BDNF was dispersed in a hydrogel composed of hyaluronan and methyl cellulose (HAMC) with poly(lactic-co-glycolic acid) (PLGA) nanoparticles and this composite was deposited epi-cortically, directly above the stroke lesion. BDNF delivery augmented plasticity, as evidenced by synaptophysin staining in the contralesional hemisphere of BDNF-treated rats, and presence of the vehicle reduced the lesion volume and prevented neuron loss in peri-lesional tissue. With local, sustained delivery directly to the brain, we demonstrate the benefit of BDNF in the treatment of stroke injury in a rodent model.

PMID: 30612516 [PubMed - as supplied by publisher]



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Initial Guidelines for Manuscripts Employing Data-independent Acquisition Mass Spectrometry for Proteomic Analysis.

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Initial Guidelines for Manuscripts Employing Data-independent Acquisition Mass Spectrometry for Proteomic Analysis.

Mol Cell Proteomics. 2019 Jan;18(1):1-2

Authors: Chalkley RJ, MacCoss MJ, Jaffe JD, Röst HL

PMID: 30602589 [PubMed - in process]



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Context-explorer: Analysis of spatially organized protein expression in high-throughput screens.

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Context-explorer: Analysis of spatially organized protein expression in high-throughput screens.

PLoS Comput Biol. 2019 01;15(1):e1006384

Authors: Ostblom J, Nazareth EJP, Tewary M, Zandstra PW

Abstract
A growing body of evidence highlights the importance of the cellular microenvironment as a regulator of phenotypic and functional cellular responses to perturbations. We have previously developed cell patterning techniques to control population context parameters, and here we demonstrate context-explorer (CE), a software tool to improve investigation cell fate acquisitions through community level analyses. We demonstrate the capabilities of CE in the analysis of human and mouse pluripotent stem cells (hPSCs, mPSCs) patterned in colonies of defined geometries in multi-well plates. CE employs a density-based clustering algorithm to identify cell colonies. Using this automatic colony classification methodology, we reach accuracies comparable to manual colony counts in a fraction of the time, both in micropatterned and unpatterned wells. Classifying cells according to their relative position within a colony enables statistical analysis of spatial organization in protein expression within colonies. When applied to colonies of hPSCs, our analysis reveals a radial gradient in the expression of the transcription factors SOX2 and OCT4. We extend these analyses to colonies of different sizes and shapes and demonstrate how the metrics derived by CE can be used to asses the patterning fidelity of micropatterned plates. We have incorporated a number of features to enhance the usability and utility of CE. To appeal to a broad scientific community, all of the software's functionality is accessible from a graphical user interface, and convenience functions for several common data operations are included. CE is compatible with existing image analysis programs such as CellProfiler and extends the analytical capabilities already provided by these tools. Taken together, CE facilitates investigation of spatially heterogeneous cell populations for fundamental research and drug development validation programs.

PMID: 30601802 [PubMed - indexed for MEDLINE]



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Targeting discriminatory SNPs in Salmonella enterica serovar Heidelberg genomes using RNase H2-dependent PCR.

Targeting discriminatory SNPs in Salmonella enterica serovar Heidelberg genomes using RNase H2-dependent PCR.

J Microbiol Methods. 2018 Dec 25;:

Authors: Labbé G, Rankin MA, Robertson J, Moffat J, Giang E, Lee LK, Ziebell K, MacKinnon J, Laing CR, Jane Parmley E, Agunos A, Daignault D, Bekal S, Chui L, MacDonald KA, Hoang L, Slavic D, Ramsay D, Pollari F, Nash JHE, Johnson RP

Abstract
We report a novel RNase H2-dependent PCR (rhPCR) genotyping assay for a small number of discriminatory single-nucleotide polymorphisms (SNPs) that identify lineages and sub-lineages of the highly clonal pathogen Salmonella Heidelberg (SH). Standard PCR primers targeting numerous SNP locations were initially designed in silico, modified to be RNase H2-compatible, and then optimized by laboratory testing. Optimization often required repeated cycling through variations in primer design, assay conditions, reagent concentrations and selection of alternative SNP targets. The final rhPCR assay uses 28 independent rhPCR reactions to target 14 DNA bases that can distinguish 15 possible lineages and sub-lineages of SH. On evaluation, the assay correctly identified the 12 lineages and sub-lineages represented in a panel of 75 diverse SH strains. Non-specific amplicons were observed in 160 (15.2%) of the 1050 reactions, but due to their low intensity did not compromise assay performance. Furthermore, in silico analysis of 500 closed genomes from 103 Salmonella serovars and laboratory rhPCR testing of five prevalent Salmonella serovars including SH indicated the assay can identify Salmonella isolates as SH, since only SH isolates generated amplicons from all 14 target SNPs. The genotyping results can be fully correlated with whole genome sequencing (WGS) data in silico. This fast and economical assay, which can identify SH isolates and classify them into related or unrelated lineages and sub-lineages, has potential applications in outbreak identification, source attribution and microbial source tracking.

PMID: 30592979 [PubMed - as supplied by publisher]



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Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

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Rationally Designed 3D Hydrogels Model Invasive Lung Diseases Enabling High-Content Drug Screening.

Adv Mater. 2018 Dec 27;:e1806214

Authors: Tam RY, Yockell-Lelièvre J, Smith LJ, Julian LM, Baker AEG, Choey C, Hasim MS, Dimitroulakos J, Stanford WL, Shoichet MS

Abstract
Cell behavior is highly dependent upon microenvironment. Thus, to identify drugs targeting metastatic cancer, screens need to be performed in tissue mimetic substrates that allow cell invasion and matrix remodeling. A novel biomimetic 3D hydrogel platform that enables quantitative analysis of cell invasion and viability at the individual cell level is developed using automated data acquisition methods with an invasive lung disease (lymphangioleiomyomatosis, LAM) characterized by hyperactive mammalian target of rapamycin complex 1 (mTORC1) signaling as a model. To test the lung-mimetic hydrogel platform, a kinase inhibitor screen is performed using tuberous sclerosis complex 2 (TSC2) hypomorphic cells, identifying Cdk2 inhibition as a putative LAM therapeutic. The 3D hydrogels mimic the native niche, enable multiple modes of invasion, and delineate phenotypic differences between healthy and diseased cells, all of which are critical to effective drug screens of highly invasive diseases including lung cancer.

PMID: 30589121 [PubMed - as supplied by publisher]



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Hypoxia-inducible factor drives vascularization of modularly assembled engineered tissue.

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Hypoxia-inducible factor drives vascularization of modularly assembled engineered tissue.

Tissue Eng Part A. 2018 Dec 26;:

Authors: Lam GC, Sefton MV

Abstract
Robust vascularization is critical for engineering tissues of clinically relevant size and cell loads. Delineating the rate limiting steps underlying vascularization is necessary to creating strategies for faster, better vascularization of tissue constructs. We used two inhibitory methods to dissect the role of hypoxia inducible factor (HIF) in vascularization-inducing engineered tissues, here constructed from self-assembly of sub-millimeter sized tissues injected subcutaneously. Both systemic pharmacological inhibition using digoxin, and genetic inhibition (shRNA-transduced endothelial cells) reduced vascularization and oxygenation within constructs, but elicited different mechanisms of action. Systemic inhibition negatively impacted early (day 3) recruitment of host-derived endothelial progenitor cells and macrophages/monocytes to the implant. Genetic inhibition in graft-derived endothelial cells impaired their survival in low serum conditions as well as their baseline angiogenic function. Together, our study demonstrates that HIF is an important driver of vascularization in tissue constructs. While hypoxia is assumed to be an important feature of tissue engineering, this study directly connects inhibition of vascularization to HIF inhibition.

PMID: 30585759 [PubMed - as supplied by publisher]



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Influencing neuroplasticity in stroke treatment with advanced biomaterials-based approaches.

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Influencing neuroplasticity in stroke treatment with advanced biomaterials-based approaches.

Adv Drug Deliv Rev. 2018 Dec 20;:

Authors: Obermeyer JM, Gracias A, Ho E, Shoichet MS

Abstract
Since the early 1990s, we have known that the adult brain is not static and has the capacity to repair itself. The delivery of various therapeutic factors and cells have resulted in some exciting pre-clinical and clinical outcomes in stroke models by targeting post-injury plasticity to enhance recovery. Developing a deeper understanding of the pathways that modulate plasticity will enable us to optimize delivery strategies for therapeutics and achieve more robust effects. Biomaterials are a key tool for the optimization of these potential treatments, owing to their biocompatibility and tunability. In this review, we identify factors and targets that impact plastic processes known to contribute to recovery, discuss the role of biomaterials in enhancing the efficacy of treatment strategies, and suggest combinatorial approaches based on the stage of injury progression.

PMID: 30579882 [PubMed - as supplied by publisher]



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The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans.

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The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans.

Elife. 2018 Dec 21;7:

Authors: Davis GM, Tu S, Anderson JWT, Colson RN, Gunzburg MJ, Francisco MA, Ray D, Shrubsole SP, Sobotka JA, Seroussi U, Lao RX, Maity T, Wu MZ, McJunkin K, Morris QD, Hughes TR, Wilce JA, Claycomb JM, Weng Z, Boag PR

Abstract
Proper regulation of germline gene expression is essential for fertility and maintaining species integrity. In the C. elegans germline, a diverse repertoire of regulatory pathways promote the expression of endogenous germline genes and limit the expression of deleterious transcripts to maintain genome homeostasis. Here we show that the conserved TRIM-NHL protein, NHL-2, plays an essential role in the C. elegans germline, modulating germline chromatin and meiotic chromosome organization. We uncover a role for NHL-2 as a co-factor in both positively (CSR-1) and negatively (HRDE-1) acting germline 22G-small RNA pathways and the somatic nuclear RNAi pathway. Furthermore, we demonstrate that NHL-2 is a bona fide RNA binding protein and, along with RNA-seq data point to a small RNA independent role for NHL-2 in regulating transcripts at the level of RNA stability. Collectively, our data implicate NHL-2 as an essential hub of gene regulatory activity in both the germline and soma.

PMID: 30575518 [PubMed - as supplied by publisher]



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