Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Digital microfluidics and nuclear magnetic resonance spectroscopy for in situ diffusion measurements and reaction monitoring.

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Digital microfluidics and nuclear magnetic resonance spectroscopy for in situ diffusion measurements and reaction monitoring.

Lab Chip. 2019 Jan 16;:

Authors: Swyer I, von der Ecken S, Wu B, Jenne A, Soong R, Vincent F, Schmidig D, Frei T, Busse F, Stronks HJ, Simpson AJ, Wheeler AR

Abstract
In recent years microcoils and related structures have been developed to increase the mass sensitivity of nuclear magnetic resonance spectroscopy, allowing this extremely powerful analytical technique to be extended to small sample volumes (<5 μl). In general, microchannels have been used to deliver the samples of interest to these microcoils; however, these systems tend to have large dead volumes and require more complex fluidic connections. Here, we introduce a two-plate digital microfluidic (DMF) strategy to interface small-volume samples with NMR microcoils. In this system, a planar microcoil is surrounded by a copper plane that serves as the counter-electrode for the digital microfluidic device, allowing for precise control of droplet position and shape. This feature allows for the user-determination of the orientation of droplets relative to the main axes of the shim stack, permitting improved shimming and a more homogeneous magnetic field inside the droplet below the microcoil, which leads to improved spectral lineshape. This, along with high-fidelity droplet actuation, allows for rapid shimming strategies (developed over decades for vertically oriented NMR tubes) to be employed, permitting the determination of reaction-product diffusion coefficients as well as quantitative monitoring of reactive intermediates. We propose that this system paves the way for new and exciting applications for in situ analysis of small samples by NMR spectroscopy.

PMID: 30648175 [PubMed - as supplied by publisher]



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Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments.

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Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments.

Nat Protoc. 2019 02;14(2):415-440

Authors: Simpkins SW, Deshpande R, Nelson J, Li SC, Piotrowski JS, Ward HN, Yashiroda Y, Osada H, Yoshida M, Boone C, Myers CL

Abstract
The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical-genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis. The software has also successfully processed data from Schizosaccharomyces pombe, Escherichia coli, and Zymomonas mobilis mutant collections. We provide general recommendations for the design of large-scale, multiplexed barcode sequencing experiments. The procedure outlined here was used to score interactions for ~4 million chemical-by-mutant combinations in our recently published chemical-genetic interaction screen of nearly 14,000 chemical compounds across seven diverse compound collections. Here we selected a representative subset of these data on which to demonstrate our analysis pipeline. BEAN-counter is open source, written in Python, and freely available for academic use. Users should be proficient at the command line; advanced users who wish to analyze larger datasets with hundreds or more conditions should also be familiar with concepts in analysis of high-throughput biological data. BEAN-counter encapsulates the knowledge we have accumulated from, and successfully applied to, our multiplexed, pooled barcode sequencing experiments. This protocol will be useful to those interested in generating their own high-dimensional, quantitative characterizations of gene or chemical function in a high-throughput manner.

PMID: 30635653 [PubMed - indexed for MEDLINE]



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Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species-dependent essential genes.

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Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species-dependent essential genes.

Genome Res. 2019 Jan 11;:

Authors: Sanchez MR, Payen C, Cheong F, Hovde BT, Bissonnette S, Arkin AP, Skerker JM, Brem RB, Caudy AA, Dunham MJ

Abstract
To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of S. cerevisiae We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with S. cerevisiae predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that trans-acting factors that are background-dependent contribute to differential gene essentiality. This study demonstrates that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotation. Our method can be widely applied in microorganisms to further our understanding of genome evolution.

PMID: 30635343 [PubMed - as supplied by publisher]



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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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