Donnelly Centre for Cellular and Biomolecular Research

PubMed

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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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Predicting changes in protein stability caused by mutation using sequence- and structure-based methods in a CAGI5 blind challenge.

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Predicting changes in protein stability caused by mutation using sequence- and structure-based methods in a CAGI5 blind challenge.

Hum Mutat. 2019 Jun 27;:

Authors: Strokach A, Corbi-Verge C, Kim PM

Abstract
Predicting the impact of mutations on proteins remains an important problem. As part of the CAGI5 frataxin challenge, we evaluate the accuracy with which Provean, FoldX, and ELASPIC can predict changes in the Gibbs free energy of a protein using a limited data set of eight mutations. We find that different methods have distinct strengths and limitations, with no method being strictly superior to other methods on all metrics. ELASPIC achieves the highest accuracy while also providing a web interface which simplifies the evaluation and analysis of mutations. FoldX is slightly less accurate than ELASPIC but is easier to run locally, as it does not depend on external tools or datasets. Provean achieves reasonable results while being computational less expensive than the other methods and not requiring a structure of the protein. In addition to methods submitted to the CAGI5 competition, and with the aim to inform about other methods with high accuracy, we also evaluate predictions made by Rosetta's ddg_monomer protocol, Rosetta's cartesian_ddg protocol, and thermodynamic integration calculations using Amber package. ELASPIC still achieves the highest accuracy, while Rosetta's catesian_ddg protocol appears to perform best in capturing the overall trend in the data. This article is protected by copyright. All rights reserved.

PMID: 31243847 [PubMed - as supplied by publisher]



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Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.

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Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.

Nat Commun. 2019 06 26;10(1):2806

Authors: Thavalingam A, Cheng Z, Garcia B, Huang X, Shah M, Sun W, Wang M, Harrington L, Hwang S, Hidalgo-Reyes Y, Sontheimer EJ, Doudna J, Davidson AR, Moraes TF, Wang Y, Maxwell KL

Abstract
CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.

PMID: 31243272 [PubMed - indexed for MEDLINE]



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Proceedings of the Canadian Frailty Network Workshop: Identifying Biomarkers of Frailty to Support Frailty Risk Assessment, Diagnosis and Prognosis. Toronto, January 15, 2018.

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Proceedings of the Canadian Frailty Network Workshop: Identifying Biomarkers of Frailty to Support Frailty Risk Assessment, Diagnosis and Prognosis. Toronto, January 15, 2018.

J Frailty Aging. 2019;8(3):106-116

Authors: Muscedere J, Kim PM, Afilalo J, Balion C, Baracos VE, Bowdish D, Cesari M, Erusalimsky JD, Fülöp T, Heckman G, Howlett SE, Khadaroo RG, Kirkland JL, Rodriguez Mañas L, Marzetti E, Paré G, Raina P, Rockwood K, Sinclair A, Skappak C, Verschoor C, Walter S

Abstract
The Canadian Frailty Network (CFN), a pan-Canadian not-for-profit organization funded by the Government of Canada through the Networks of Centres of Excellence Program, is dedicated to improving the care of older Canadians living with frailty. The CFN has partnered with the Canadian Longitudinal Study on Aging (CLSA) to measure potential frailty biomarkers in biological samples (whole blood, plasma, urine) collected in over 30,000 CLSA participants. CFN hosted a workshop in Toronto on January 15 2018, bringing together experts in the field of biomarkers, aging and frailty. The overall objectives of the workshop were to start building a consensus on potential frailty biomarker domains and identify specific frailty biomarkers to be measured in the CLSA biological samples. The workshop was structured with presentations in the morning to frame the discussions for the afternoon session, which was organized as a free-flowing discussion to benefit from the expertise of the participants. Participants and speakers were from Canada, Italy, Spain, United Kingdom and the United States. Herein we provide pertinent background information, a summary of all the presentations with key figures and tables, and the distillation of the discussions. In addition, moving forward, the principles CFN will use to approach frailty biomarker research and development are outlined. Findings from the workshop are helping CFN and CLSA plan and conduct the analysis of biomarkers in the CLSA samples and which will inform a follow-up data access competition.

PMID: 31237310 [PubMed - indexed for MEDLINE]



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Rhodoquinone biosynthesis in C.elegans requires precursors generated by the kynurenine pathway.

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Rhodoquinone biosynthesis in C.elegans requires precursors generated by the kynurenine pathway.

Elife. 2019 Jun 24;8:

Authors: Del Borrello S, Lautens M, Dolan K, Tan JH, Davie T, Schertzberg MR, Spensley MA, Caudy AA, Fraser AG

Abstract
Parasitic helminths infect over a billion humans. To survive in the low oxygen environment of their hosts, these parasites use unusual anaerobic metabolism - this requires rhodoquinone (RQ), an electron carrier that is made by very few animal species. Crucially RQ is not made or used by any parasitic hosts and RQ synthesis is thus an ideal target for anthelmintics. However, little is known about how RQ is made and no drugs are known to block RQ synthesis. C.elegans makes RQ and can use RQ-dependent metabolic pathways - here, we use C.elegans genetics to show that tryptophan degradation via the kynurenine pathway is required to generate the key amine-containing precursors for RQ synthesis. We show that C.elegans requires RQ for survival in hypoxic conditions and, finally, we establish a high throughput assay for drugs that block RQ-dependent metabolism. This may drive the development of a new class of anthelmintic drugs. This study is a key first step in understanding how RQ is made in parasitic helminths.

PMID: 31232688 [PubMed - as supplied by publisher]



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Optimization of peptidic HIV-1 fusion inhibitor T20 by phage display.

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Optimization of peptidic HIV-1 fusion inhibitor T20 by phage display.

Protein Sci. 2019 Jun 22;:

Authors: Chen G, Cook JD, Ye W, Lee JE, Sidhu SS

Abstract
The HIV fusion inhibitor T20 has been approved to treat those living with HIV/AIDS, but treatment gives rise to resistant viruses. Using combinatorial phage-displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five-helix bundle (5HB) mimetic of HIV-1 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 to 5HB. The scan of T20 revealed that 12 of its 36 positions were conserved for 5HB binding, which cluster into three epitopes: hydrophobic epitopes at the ends and a central dyad of hydrophilic residues. The scan also revealed that the T20 sequence was highly adaptable to mutations at most positions, demonstrating a striking structural plasticity that allows multiple amino acid substitutions at contact points to adapt to conformational changes, and also at non-contact points to fine-tune the interface. Based on the scan result and structural knowledge of the gp41 fusion intermediate, a library was designed with tailored diversity at particular positions of T20 and was used to derive a variant (T20v1) that was found to be a highly effective inhibitor of infection by multiple HIV-1 variants, including a common T20-escape mutant. These findings show that the plasticity of the T20 functional sequence space can be exploited to develop variants that overcome resistance of HIV-1 variants to T20 itself, and demonstrate the utility of saturation scanning for rapid epitope mapping and protein engineering. This article is protected by copyright. All rights reserved.

PMID: 31228294 [PubMed - as supplied by publisher]



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