Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

The optoelectronic microrobot: A versatile toolbox for micromanipulation.

The optoelectronic microrobot: A versatile toolbox for micromanipulation.

Proc Natl Acad Sci U S A. 2019 Jul 09;:

Authors: Zhang S, Scott EY, Singh J, Chen Y, Zhang Y, Elsayed M, Chamberlain MD, Shakiba N, Adams K, Yu S, Morshead CM, Zandstra PW, Wheeler AR

Abstract
Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of "load," "transport," and "deliver," which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell-cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond.

PMID: 31289234 [PubMed - as supplied by publisher]



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Assessing micrometastases as a target for nanoparticles using 3D microscopy and machine learning.

Assessing micrometastases as a target for nanoparticles using 3D microscopy and machine learning.

Proc Natl Acad Sci U S A. 2019 Jul 08;:

Authors: Kingston BR, Syed AM, Ngai J, Sindhwani S, Chan WCW

Abstract
Metastasis of solid tumors is a key determinant of cancer patient survival. Targeting micrometastases using nanoparticles could offer a way to stop metastatic tumor growth before it causes excessive patient morbidity. However, nanoparticle delivery to micrometastases is difficult to investigate because micrometastases are small in size and lie deep within tissues. Here, we developed an imaging and image analysis workflow to analyze nanoparticle-cell interactions in metastatic tumors. This technique combines tissue clearing and 3D microscopy with machine learning-based image analysis to assess the physiology of micrometastases with single-cell resolution and quantify the delivery of nanoparticles within them. We show that nanoparticles access a higher proportion of cells in micrometastases (50% nanoparticle-positive cells) compared with primary tumors (17% nanoparticle-positive cells) because they reside close to blood vessels and require a small diffusion distance to reach all tumor cells. Furthermore, the high-throughput nature of our image analysis workflow allowed us to profile the physiology and nanoparticle delivery of 1,301 micrometastases. This enabled us to use machine learning-based modeling to predict nanoparticle delivery to individual micrometastases based on their physiology. Our imaging method allows researchers to measure nanoparticle delivery to micrometastases and highlights an opportunity to target micrometastases with nanoparticles. The development of models to predict nanoparticle delivery based on micrometastasis physiology could enable personalized treatments based on the specific physiology of a patient's micrometastases.

PMID: 31285340 [PubMed - as supplied by publisher]



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Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen.

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Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen.

PLoS Biol. 2019 Jul 08;17(7):e3000358

Authors: O'Meara TR, O'Meara MJ, Polvi EJ, Pourhaghighi MR, Liston SD, Lin ZY, Veri AO, Emili A, Gingras AC, Cowen LE

Abstract
Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein-protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans. In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules.

PMID: 31283755 [PubMed - as supplied by publisher]



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Assessing predictions on fitness effects of missense variants in calmodulin.

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Assessing predictions on fitness effects of missense variants in calmodulin.

Hum Mutat. 2019 Jul 08;:

Authors: Zhang J, Kinch LN, Cong Q, Katsonis P, Lichtarge O, Savojardo C, Babbi G, Martelli PL, Capriotti E, Casadio R, Garg A, Pal D, Weile J, Sun S, Verby M, Roth FP, Grishin NV

Abstract
This paper reports the evaluation of predictions for the "CALM1" challenge in the 5th round of the Critical Assessment of Genome Interpretation held in 2018. In the challenge, the participants were asked to predict effects on yeast growth caused by missense variants of human calmodulin, a highly conserved protein in eukaryotic cells sensing calcium concentration. The performance of predictors implementing different algorithms and methods is similar. Most predictors are able to identify the deleterious or benign variants with modest accuracy, with a baseline predictor based purely on sequence conservation slightly outperforming the submitted predictions. Nevertheless, we think that the accuracy of predictions remains far from satisfactory, and the field awaits substantial improvements. The most poorly predicted variants in this round surround functional CALM1 sites that bind calcium or peptide, which suggests that better incorporation of structural analysis may help improve predictions. This article is protected by copyright. All rights reserved.

PMID: 31283071 [PubMed - as supplied by publisher]



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The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation.

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The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation.

Curr Biol. 2019 Jun 28;:

Authors: Garg J, Saettone A, Nabeel-Shah S, Cadorin M, Ponce M, Marquez S, Pu S, Greenblatt J, Lambert JP, Pearlman RE, Fillingham J

Abstract
Mediator is a large protein complex required for basal and regulated expression of most RNA polymerase II (RNAP II)-transcribed genes, in part due to its interaction with and phosphorylation of the conserved C-terminal domain (CTD) of Rpb1 [1, 2]. Mediator has been implicated in many aspects of gene expression including chromatin looping [3], higher-order chromatin folding [4], mRNA processing [5] and export [6], and transcriptional memory [7]. Mediator is thought to have played a major role during eukaryotic diversification [8, 9], although its function remains unknown in evolutionarily deep branching eukaryotes lacking canonical CTD heptad repeats. We used the ciliate protozoan Tetrahymena thermophila as a model organism whose genome encodes a highly divergent Rpb1 lacking canonical CTD heptad repeats. We endogenously tagged the Med31 subunit of the Mediator complex and performed affinity purification coupled with mass spectrometry (AP-MS) to identify Mediator subunits. We found that Med31 physically interacts with a large number of proteins (>20), several of which share similarities to canonical Mediator subunits in yeast and humans as well as Tetrahymena-specific proteins. Furthermore, Med31 ChIP-seq analysis suggested a global role for Mediator in transcription regulation. We demonstrated that MED31 knockdown in growing Tetrahymena results in the ectopic expression of developmental genes important for programmed DNA rearrangements. In addition, indirect immunofluorescence revealed Med31 localization in meiotic micronuclei, implicating Mediator in RNAPII-dependent ncRNA transcription. Our results reveal structural and functional insights and implicate Mediator as an ancient cellular machinery for transcription regulation with a possible involvement in global transcription of ncRNAs.

PMID: 31280994 [PubMed - as supplied by publisher]



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Target 2035: probing the human proteome.

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Target 2035: probing the human proteome.

Drug Discov Today. 2019 Jul 03;:

Authors: Carter AJ, Kraemer O, Zwick M, Mueller-Fahrnow A, Arrowsmith CH, Edwards AM

Abstract
Biomedical scientists tend to focus on only a small fraction of the proteins encoded by the human genome despite overwhelming genetic evidence that many understudied proteins are important for human disease. One of the best ways to interrogate the function of a protein and to determine its relevance as a drug target is by using a pharmacological modulator, such as a chemical probe or an antibody. If these tools were available for most human proteins, it should be possible to translate the tremendous advances in genomics into a greater understanding of human health and disease, and catalyze the creation of innovative new medicines. Target 2035 is a global federation for developing and applying new technologies with the goal of creating chemogenomic libraries, chemical probes, and/or functional antibodies for the entire proteome.

PMID: 31278990 [PubMed - as supplied by publisher]



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Supervised Learning and Mass Spectrometry Predicts the in Vivo Fate of Nanomaterials.

Supervised Learning and Mass Spectrometry Predicts the in Vivo Fate of Nanomaterials.

ACS Nano. 2019 Jul 03;:

Authors: Lazarovits J, Sindhwani S, Tavares AJ, Zhang Y, Song F, Audet J, Krieger JR, Syed AM, Stordy B, Chan WCW

Abstract
The surface of nanoparticles changes immediately after intravenous injection because blood proteins adsorb on the surface. How this interface changes during circulation and its impact on nanoparticle distribution within the body is not understood. Here, we developed a workflow to show that the evolution of proteins on nanoparticle surfaces predicts the biological fate of nanoparticles in vivo. This workflow involves extracting nanoparticles at multiple time points from circulation, isolating the proteins off the surface and performing proteomic mass spectrometry. The mass spectrometry protein library served as inputs, while blood clearance and organ accumulation were used as outputs to train a supervised deep neural network that predicts nanoparticle biological fate. In a double-blinded study, we tested the network by predicting nanoparticle spleen and liver accumulation with upward of 94% accuracy. Our neural network discovered that the mechanism of liver and spleen uptake is due to patterns of a multitude of nanoparticle surface adsorbed proteins. There are too many combinations to change these proteins manually using chemical or biological inhibitors to alter clearance. Therefore, we developed a technique that uses the host to act as a bioreactor to prepare nanoparticles with predictable clearance patterns that reduce liver and spleen uptake by 50% and 70%, respectively. These techniques provide opportunities to both predict nanoparticle behavior and also to engineer surface chemistries that are specifically designed by the body.

PMID: 31268684 [PubMed - as supplied by publisher]



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The Blood Compatibility Challenge: Editorial introduction.

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The Blood Compatibility Challenge: Editorial introduction.

Acta Biomater. 2019 Jun 25;:

Authors: Sefton MV, Sperling C, Maitz MF, Werner C

PMID: 31252174 [PubMed - as supplied by publisher]



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Ion-Exchange Based Immobilization of Chromogenic Reagents on Microfluidic Paper Analytical Devices.

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Ion-Exchange Based Immobilization of Chromogenic Reagents on Microfluidic Paper Analytical Devices.

Anal Chem. 2019 Jun 28;:

Authors: Rahbar M, Wheeler AR, Paull B, Macka M

Abstract
Distance-based detection methods, as used in development of microfluidic paper analytical devices (μPADs), rely on the dynamic formation of a colored band along the length of the paper microfluidic channels. The color change is driven by the reaction of chromogenic reagents (typically water-insoluble) that are bound to the paper, thus not subject to being washed away by the sample flow along the detection channel. Here, we introduce the use of an anion-exchange filter paper (as a replacement for standard, unmodified filter paper) for distance-based detection in μPADs, in order to immobilize the water-soluble anionic reagents upon the paper detection channels based on ion-exchange interactions of the oppositely charged paper (protonated tertiary amine groups) and the anionic groups of the reagents. The ion-exchange (IE) paper was initially characterized and its properties were compared with standard cellulose paper. The IE paper was shown to be capable of strong retention of anionic reagents exhibiting acidic functional groups (carboxylic, sulfonic), which become deprotonated and negatively charged when in contact with the IE paper. The effect of the ionic strength (10-250 mM Cl-) and pH (1-13) on the immobilization of the investigated reagents were also determined. The IE-μPADs were then modified with anionic chromogenic reagents and applied to distance-based determination of total calcium (LOD = 0.03 mM) and total acidity (LOD = 2.5 mM) content in serum and wine samples, respectively. The detailed mechanisms of the developed assays on the IE paper are also discussed. We propose that IE-μPADs represent a useful new addition to the distance-based detection toolbox and considerably enhance the applicability of such a detection method.

PMID: 31251584 [PubMed - as supplied by publisher]



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Triggered Release Enhances the Cytotoxicity of Stable Colloidal Drug Aggregates.

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Triggered Release Enhances the Cytotoxicity of Stable Colloidal Drug Aggregates.

ACS Chem Biol. 2019 Jun 25;:

Authors: Donders EN, Ganesh AN, Torosyan H, Lak P, Shoichet BK, Shoichet MS

Abstract
Chemotherapeutics that self-assemble into colloids have limited efficacy above their critical aggregation concentration due to their inability to penetrate intact plasma membranes. Even when colloid uptake is promoted, issues with colloid escape from the endolysosomal pathway persist. By stabilizing acid-responsive lapatinib colloids through coaggregation with fulvestrant, and inclusion of transferrin, we demonstrate colloid internalization by cancer cells, where subsequent lapatinib ionization leads to endosomal leakage and increased cytotoxicity. These results demonstrate a strategy for triggered drug release from stable colloidal aggregates.

PMID: 31243955 [PubMed - as supplied by publisher]



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