Donnelly Centre for Cellular and Biomolecular Research

PubMed

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Engineering Steps for Mobile Point-of-Care Diagnostic Devices.

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Engineering Steps for Mobile Point-of-Care Diagnostic Devices.

Acc Chem Res. 2019 Aug 20;:

Authors: Malekjahani A, Sindhwani S, Syed AM, Chan WCW

Abstract
Mobile phone technology is a perfect companion for point-of-care diagnostics as they come equipped with advanced processors, high resolution cameras, and network connectivity. Despite several academic pursuits, only a few mobile phone diagnostics have been tested in the field, commercialized or achieved regulatory approval. This review will address the challenges associated with developing mobile diagnostics and suggest strategies to overcome them. We aim to provide a resource for researchers to accelerate the development of new diagnostics. Our Account includes an overview of published mobile phone diagnostics and highlights lessons learned from their approach to diagnostic development. Also, we have included recommendations from regulatory and public health agencies, such as the U.S. Food and Drug Administration and World Health Organization, to further guide researchers. We believe that the development of mobile phone point-of-care diagnostics takes place in four distinct steps: (1) Needs and Value Assessment, (2) Technology Development, (3) Preclinical Verification, and (4) Clinical Validation and Field Trials. During each step, we outline developmental strategies to help researchers avoid potential challenges. (1) Researchers commonly develop devices to maximize technical parameters such as sensitivity and time which do not necessarily translate to increased clinical impact. Researchers must focus on assessing specific diagnostic needs and the value which a potential device would offer. (2) Often, researchers claim they have developed devices for feasible implementation at the point-of-care, yet they rely on laboratory resources. Researchers must develop equipment-free devices which are agnostic to any mobile phone. (3) Another challenge researchers face is decreased performance during field evaluations relative to initial laboratory verification. Researchers must ensure that they simulate the field conditions during laboratory verification to achieve successful translation. (4) Finally, proper field testing of devices must be performed in conditions which match that of the final intended use. The future of mobile phone point-of-care diagnostic devices is bright and has the potential to radically change how patients are diagnosed. Before we reach this point, researchers must take a step backward and focus on the first-principles of basic research. The widespread adoption and rapid scaling of these devices can only be achieved once the fundamentals have been considered. The insights and strategies provided here will help researchers avoid pitfalls, streamline development and make better decisions during the development of new diagnostics. Further, we believe this Account can help push the field of mobile diagnostics toward increased productivity, leading to more approved devices and ultimately helping curb the burden of disease worldwide.

PMID: 31430118 [PubMed - as supplied by publisher]



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Post-replication repair: Rad5/HLTF regulation, activity on undamaged templates, and relationship to cancer.

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Post-replication repair: Rad5/HLTF regulation, activity on undamaged templates, and relationship to cancer.

Crit Rev Biochem Mol Biol. 2019 Aug 20;:1-32

Authors: Gallo D, Brown GW

Abstract
The eukaryotic post-replication repair (PRR) pathway allows completion of DNA replication when replication forks encounter lesions on the DNA template and are mediated by post-translational ubiquitination of the DNA sliding clamp proliferating cell nuclear antigen (PCNA). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases to replicate past DNA lesions in an error-prone manner while addition of K63-linked polyubiquitin chains signals for error-free template switching to the sister chromatid. Central to both branches is the E3 ubiquitin ligase and DNA helicase Rad5/helicase-like transcription factor (HLTF). Mutations in PRR pathway components lead to genomic rearrangements, cancer predisposition, and cancer progression. Recent studies have challenged the notion that the PRR pathway is involved only in DNA lesion tolerance and have shed new light on its roles in cancer progression. Molecular details of Rad5/HLTF recruitment and function at replication forks have emerged. Mounting evidence indicates that PRR is required during lesion-less replication stress, leading to TLS polymerase activity on undamaged templates. Analysis of PRR mutation status in human cancers and PRR function in cancer models indicates that down regulation of PRR activity is a viable strategy to inhibit cancer cell growth and reduce chemoresistance. Here, we review these findings, discuss how they change our views of current PRR models, and look forward to targeting the PRR pathway in the clinic.

PMID: 31429594 [PubMed - as supplied by publisher]



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A 3D Printed Device for Low Cost Neural Stimulation in Mice.

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A 3D Printed Device for Low Cost Neural Stimulation in Mice.

Front Neurosci. 2019;13:784

Authors: Morrison TJ, Sefton E, Marquez-Chin M, Popovic MR, Morshead CM, Naguib HE

Abstract
Electrical stimulation of the brain through the implantation of electrodes is an effective treatment for certain diseases and the focus of a large body of research investigating new cell mechanisms, neurological phenomena, and treatments. Electrode devices developed for stimulation in rodents vary widely in size, cost, and functionality, with the majority of recent studies presenting complex, multi-functional designs. While some experiments require these added features, others are in greater need of reliable, low cost, and readily available devices that will allow surgeries to be scheduled and completed without delay. In this work, we utilize 3D printing and common electrical hardware to produce an effective 2-channel stimulation device that meets these requirements. Our stimulation electrode has not failed in over 60 consecutive surgeries, costs less than $1 USD, and can be assembled in less than 20 min. 3D printing minimizes the amount of material used in manufacturing the device and enables one to match the curvature of the connector's base with the curvature of the mouse skull, producing an ultra-lightweight, low size device with improved adhesion to the mouse skull. The range of the stimulation parameters used with the proposed device was: pulse amplitude 1-200 μA, pulse duration 50-500 μs and pulse frequency 1-285 Hz.

PMID: 31417347 [PubMed]



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Methacrylic Acid Copolymer Coating Enhances Constructive Remodeling of Polypropylene Mesh by Increasing the Vascular Response.

Methacrylic Acid Copolymer Coating Enhances Constructive Remodeling of Polypropylene Mesh by Increasing the Vascular Response.

Adv Healthc Mater. 2019 Aug 13;:e1900667

Authors: Coindre VF, Carleton MM, Sefton MV

Abstract
This study reports that a methacrylic acid (MAA)-based copolymer coating generates constructive remodeling of polypropylene (PP) surgical mesh in a subcutaneous model. This coating is non-bioresorbable and follows the architecture of the mesh without impeding connective tissue integration. Following implantation, the tissue response is biased toward vascularization instead of fibrosis. The vessel density around the MAA mesh is double that of the uncoated mesh two weeks after implantation. This initial vasculature regresses after two weeks while mature vessels remain, suggesting an enhanced healing response. Concurrently, the MAA coating alters the foreign body response to the mesh. Fewer infiltrating cells, macrophages, and foreign body giant cells are found at the tissue-material interface three weeks after implantation. The coating also dampens inflammation, with lower expression levels of pro-inflammatory and fibrogenic signals (e.g., Tgf-β1, Tnf-α, and Il1-β) and similar expression levels of anti-inflammatory cytokines (e.g., Il10 and Il6) compared to the uncoated mesh. Contrary to other coatings that aim to mitigate the foreign body response to PP mesh, a MAA coating does not require the addition of any biological agents to have an effect, making the coated mesh an attractive candidate for soft tissue repair.

PMID: 31407481 [PubMed - as supplied by publisher]



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A receptor tyrosine kinase plays separate roles in sensory integration and associative learning in C. elegans.

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A receptor tyrosine kinase plays separate roles in sensory integration and associative learning in C. elegans.

eNeuro. 2019 Aug 01;:

Authors: Wolfe GS, Tong VW, Povse E, Merritt DM, Stegeman GW, Flibotte S, van der Kooy D

Abstract
Associative learning and sensory integration are two behavioral processes that involve the sensation and processing of stimuli followed by an altered behavioral response to these stimuli, with learning requiring memory formation and retrieval. We found that the cellular and molecular actions of scd-2 dissociate sensory integration and associative learning. This was discovered through investigation of a Caenorhabditis elegans mutation (lrn-2 (mm99)) affecting both processes. After mapping and sequencing, lrn-2 was found to be allelic to the gene, scd-2 scd-2 mediated associative learning and sensory integration operate in separate neurons as separate processes. We also find that memories can form from associations that are processed and stored independently from the integration of stimuli preceding an immediate behavioral decision.Significance Statement We show that the mutation lrn-2, a learning mutant derived from a random mutagenesis screen is allelic to scd-2, a receptor tyrosine kinase. Differences in the role of scd-2 provide the first evidence for genetic, cellular, and behavioral dissociations of sensory integration and associative learning in C. elegans We show that scd-2 uses different genetic and neuronal pathways for its role in sensory integration versus associative learning. Furthermore, this dissociation shows that sensory integration and associative learning are separate phenomena and that memories can form from associations independent of initial sensory integration. This implies memory formation can be separated from real-time sensory perception.

PMID: 31371455 [PubMed - as supplied by publisher]



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Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site.

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Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site.

Proc Natl Acad Sci U S A. 2019 Jul 26;:

Authors: Watson ER, Grace CRR, Zhang W, Miller DJ, Davidson IF, Prabu JR, Yu S, Bolhuis DL, Kulko ET, Vollrath R, Haselbach D, Stark H, Peters JM, Brown NG, Sidhu SS, Schulman BA

Abstract
Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub's C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at "exosites." However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.

PMID: 31350353 [PubMed - as supplied by publisher]



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Quantifying immune-based counterselection of somatic mutations.

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Quantifying immune-based counterselection of somatic mutations.

PLoS Genet. 2019 Jul;15(7):e1008227

Authors: Yang F, Kim DK, Nakagawa H, Hayashi S, Imoto S, Stein L, Roth FP

Abstract
Somatic mutations in protein-coding regions can generate 'neoantigens' causing developing cancers to be eliminated by the immune system. Quantitative estimates of the strength of this counterselection phenomenon have been lacking. We quantified the extent to which somatic mutations are depleted in peptides that are predicted to be displayed by major histocompatibility complex (MHC) class I proteins. The extent of this depletion depended on expression level of the neoantigenic gene, and on whether the patient had one or two MHC-encoding alleles that can display the peptide, suggesting MHC-encoding alleles are incompletely dominant. This study provides an initial quantitative understanding of counter-selection of identifiable subclasses of neoantigenic somatic variation.

PMID: 31344031 [PubMed - in process]



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Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation.

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Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation.

J Virol. 2019 Jul 24;:

Authors: Siddiqi UZ, Vaidya AS, Li X, Marcon E, Tsao SW, Greenblatt J, Frappier L

Abstract
Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication. Lytic reactivation starts with derepression of the Zp promoter controlling BZLF1 gene expression, which binds and is activated by the c-Jun transcriptional activator. Here we identified the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation. Silencing of ARKL1 in the context of EBV-positive gastric carcinoma (AGS), nasopharyngeal carcinoma (NPC43) and B cells (M81) led to increased lytic protein expression, whereas overexpression inhibited BZLF1 expression. Similar effects of ARKL1 modulation were seen on BZLF1 transcripts as well as on Zp activity in Zp reporter assays, showing ARKL1 repressed Zp. Proteomic profiling of ARKL1-host interactions identified c-Jun as an ARKL1 interactor, and reporter assays for Jun transcriptional activity showed that ARKL1 inhibited Jun activity. The ARKL1-Jun interaction required ARKL1 sequences that we previously showed mediated binding to the CK2 kinase regulatory subunit, CK2β, suggesting that CK2β might mediate the ARKL1-Jun interaction. This model was supported by the findings that silencing of CK2β, but not the CK2α catalytic subunit, abrogated the ARKL1-Jun interaction and phenocopied ARKL1 silencing in promoting EBV reactivation. Additionally, ARKL1 associated with Zp in reporter assays and this was increased by additional CK2β. Together the data indicate that ARKL1 is a negative regulator of Zp and EBV reactivation that acts by inhibiting Jun activity through a CK2β-mediated interaction.IMPORTANCE Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication and is associated with several types of cancer. We have identified a cellular protein (ARKL1) that acts to repress the reactivation of EBV from the latent to the lytic cycle. We show that ARKL1 acts to repress transcription of the EBV lytic switch protein by inhibiting the activity of the cellular transcription factor, c-Jun. This not only provides a new mechanism of regulating EBV reactivation but also identifies a novel cellular function of ARKL1 as an inhibitor of Jun-mediated transcription.

PMID: 31341047 [PubMed - as supplied by publisher]



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Benchmarking to the Gold Standard: Hyaluronan-Oxime Hydrogels Recapitulate Xenograft Models with In Vitro Breast Cancer Spheroid Culture.

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Benchmarking to the Gold Standard: Hyaluronan-Oxime Hydrogels Recapitulate Xenograft Models with In Vitro Breast Cancer Spheroid Culture.

Adv Mater. 2019 Jul 19;:e1901166

Authors: Baker AEG, Bahlmann LC, Tam RY, Liu JC, Ganesh AN, Mitrousis N, Marcellus R, Spears M, Bartlett JMS, Cescon DW, Bader GD, Shoichet MS

Abstract
Many 3D in vitro models induce breast cancer spheroid formation; however, this alone does not recapitulate the complex in vivo phenotype. To effectively screen therapeutics, it is urgently needed to validate in vitro cancer spheroid models against the gold standard of xenografts. A new oxime-crosslinked hyaluronan (HA) hydrogel is designed, manipulating gelation rate and mechanical properties to grow breast cancer spheroids in 3D. This HA-oxime breast cancer model maintains the gene expression profile most similar to that of tumor xenografts based on a pan-cancer gene expression profile (comprising 730 genes) of three different human breast cancer subtypes compared to Matrigel or conventional 2D culture. Differences in gene expression between breast cancer cultures in HA-oxime versus Matrigel or 2D are confirmed for 12 canonical pathways by gene set variation analysis. Importantly, drug response is dependent on the culture method. Breast cancer cells respond better to the Rac inhibitor (EHT-1864) and the PI3K inhibitor (AZD6482) when cultured in HA-oxime versus Matrigel. This study demonstrates the superiority of an HA-based hydrogel as a platform for in vitro breast cancer culture of both primary, patient-derived cells and cell lines, and provides a hydrogel culture model that closely matches that in vivo.

PMID: 31322299 [PubMed - as supplied by publisher]



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EPIC: software toolkit for elution profile-based inference of protein complexes.

EPIC: software toolkit for elution profile-based inference of protein complexes.

Nat Methods. 2019 Jul 15;:

Authors: Hu LZ, Goebels F, Tan JH, Wolf E, Kuzmanov U, Wan C, Phanse S, Xu C, Schertzberg M, Fraser AG, Bader GD, Emili A

Abstract
Protein complexes are key macromolecular machines of the cell, but their description remains incomplete. We and others previously reported an experimental strategy for global characterization of native protein assemblies based on chromatographic fractionation of biological extracts coupled to precision mass spectrometry analysis (chromatographic fractionation-mass spectrometry, CF-MS), but the resulting data are challenging to process and interpret. Here, we describe EPIC (elution profile-based inference of complexes), a software toolkit for automated scoring of large-scale CF-MS data to define high-confidence multi-component macromolecules from diverse biological specimens. As a case study, we used EPIC to map the global interactome of Caenorhabditis elegans, defining 612 putative worm protein complexes linked to diverse biological processes. These included novel subunits and assemblies unique to nematodes that we validated using orthogonal methods. The open source EPIC software is freely available as a Jupyter notebook packaged in a Docker container ( https://hub.docker.com/r/baderlab/bio-epic/ ).

PMID: 31308550 [PubMed - as supplied by publisher]



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