Donnelly Centre for Cellular and Biomolecular Research

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"Plug-n-Play" Sensing with Digital Microfluidics.

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"Plug-n-Play" Sensing with Digital Microfluidics.

Anal Chem. 2019 Jan 24;:

Authors: de Campos RPS, Rackus DG, Shih R, Zhao C, Liu X, Wheeler AR

Abstract
Digital microfluidics (DMF) is a platform that enables highly reconfigurable and automated fluidic operations using a generic device architecture. A unique hallmark of DMF is its "flexibility": a generic device design can be used and reused for many different, divergent fluidic operations. The flexibility of DMF is compromised when devices are permanently modified with embedded sensors. Here we introduce a solution to the "flexibility gap" between fluidic operations in digital microfluidics and embedded sensors: "plug-n-play DMF" (PnP-DMF). In PnP-DMF, devices are designed to allow for rapid and seamless exchange of sensors depending on the application needs. This paper provides "proof of concept" for PnP-DMF using commercial biosensors for glucose and β-ketone, a custom paper-based electrochemical sensor for lactate, and a generic screen-printed electroanalytical cell. We demonstrate that hot-swapping sensors between experiments allows for convenient implementation of complex processes such as automated analysis of blood samples by standard addition. Finally, we explored the suitability for using PnP sensors in tandem with other sensing modalities, combining biosensor-based electrochemical measurement of glucose with a chemiluminescent magnetic bead-based sandwich immunoassay for insulin. The latter is notable, as it constitutes the first report of an analysis of different analytes in both the supernatant and precipitate from a single sample-aliquot in a microfluidic device. The results presented here highlight the versatility of PnP-DMF, illustrating how it may be useful for a wide range of applications in diagnostics and beyond.

PMID: 30676737 [PubMed - as supplied by publisher]



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Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap.

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Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap.

Nat Protoc. 2019 02;14(2):482-517

Authors: Reimand J, Isserlin R, Voisin V, Kucera M, Tannus-Lopes C, Rostamianfar A, Wadi L, Meyer M, Wong J, Xu C, Merico D, Bader GD

Abstract
Pathway enrichment analysis helps researchers gain mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. We explain the procedures of pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome-sequencing experiments. The protocol comprises three major steps: definition of a gene list from omics data, determination of statistically enriched pathways, and visualization and interpretation of the results. We describe how to use this protocol with published examples of differentially expressed genes and mutated cancer genes; however, the principles can be applied to diverse types of omics data. The protocol describes innovative visualization techniques, provides comprehensive background and troubleshooting guidelines, and uses freely available and frequently updated software, including g:Profiler, Gene Set Enrichment Analysis (GSEA), Cytoscape and EnrichmentMap. The complete protocol can be performed in ~4.5 h and is designed for use by biologists with no prior bioinformatics training.

PMID: 30664679 [PubMed - indexed for MEDLINE]



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A web application and service for imputing and visualizing missense variant effect maps.

A web application and service for imputing and visualizing missense variant effect maps.

Bioinformatics. 2019 Jan 14;:

Authors: Yingzhou W, Weile J, Cote A, Sun S, Knapp J, Verby M, Roth FP

Abstract
Motivation: The promise of personalized genomic medicine depends on our ability to assess the functional impact of rare sequence variation. Multiplexed assays of variant effect experimentally measure the functional impact of missense variants on a massive scale. However, even after such an assay, many missense variants remain poorly measured. Here we describe a software pipeline and application to impute missing information in experimentally determined variant effect maps.
Availability: http://impute.varianteffect.org source code: https://github.com/joewuca/imputation.
Supplementary information: Supplementary data are available at Bioinformatics online.

PMID: 30649215 [PubMed - as supplied by publisher]



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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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