Donnelly Centre for Cellular and Biomolecular Research

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Recent Publications

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 - in process]



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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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The blood compatibility challenge Part 3: Material associated activation of blood cascades and cells.

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The blood compatibility challenge Part 3: Material associated activation of blood cascades and cells.

Acta Biomater. 2019 Jun 18;:

Authors: Gorbet M, Sperling C, Maitz MF, Siedleck CA, Werner C, Sefton MV

Abstract
Following protein adsorption/activation which is the first step after the contact of material surfaces and whole blood (part 2), fibrinogen is converted to fibrin and platelets become activated and assembled in the form of a thrombus. This thrombus formation is the key feature that needs to be minimized in the creation of materials with low thrombogenicity. Further aspects of blood compatibility that are important on their own are complement and leukocyte activation which are also important drivers of thrombus formation. Hence this chapter summarizes the state of knowledge on all of these cascades and cells and their interactions. For each cascade or cell type, the chapter distinguishes that which is in widespread agreement from what there is less of a consensus. STATEMENT OF SIGNIFICANCE: This paper is part 3 of a series of 4 reviews discussing the problem of biomaterial associated thrombogenicity. The objective was to highlight features of broad agreement and provide commentary on those aspects of the problem that were subject to dispute. We hope that future investigators will update these reviews as new scholarship resolves the uncertainties of today.

PMID: 31226478 [PubMed - as supplied by publisher]



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The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay.

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The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay.

Elife. 2019 Jun 20;8:

Authors: D'Orazio KN, Wu CC, Sinha NK, Loll-Krippleber R, Brown GW, Green R

Abstract
Translation of problematic sequences in mRNAs leads to ribosome collisions that trigger a series of quality control events including ribosome rescue, degradation of the stalled nascent polypeptide via the Ribosome-mediated Quality control Complex (RQC), and targeting of the mRNA for decay (No Go Decay or NGD). Previous studies provide strong evidence for the existence of an endonuclease involved in the process of NGD, though the identity of the endonuclease and the extent to which it contributes to mRNA decay remain unknown. Using a reverse genetic screen in yeast, we identify Cue2 as the conserved endonuclease that is recruited to stalled ribosomes to promote NGD. Ribosome profiling and biochemistry provide strong evidence that Cue2 cleaves mRNA within the A site of the colliding ribosome. We demonstrate that NGD primarily proceeds via Xrn1-mediated exonucleolytic decay and Cue2-mediated endonucleolytic decay normally constitutes a secondary decay pathway. Finally, we show that the Cue2-dependent pathway becomes a major contributor to NGD in cells depleted of factors required for the resolution of stalled ribosome complexes (the RQT factors including Slh1). Together these results provide insights into how multiple decay processes converge to process problematic mRNAs in eukaryotic cells.​.

PMID: 31219035 [PubMed - as supplied by publisher]



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Towards reliable named entity recognition in the biomedical domain.

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Towards reliable named entity recognition in the biomedical domain.

Bioinformatics. 2019 Jun 20;:

Authors: Giorgi JM, Bader GD

Abstract
MOTIVATION: Automatic biomedical named entity recognition (BioNER) is a key task in biomedical information extraction (IE). For some time, state-of-the-art BioNER has been dominated by machine learning methods, particularly conditional random fields (CRFs), with a recent focus on deep learning. However, recent work has suggested that the high performance of CRFs for BioNER may not generalize to corpora other than the one it was trained on. In our analysis, we find that a popular deep learning-based approach to BioNER, known as bidirectional long short-term memory network-conditional random field (BiLSTM-CRF), is correspondingly poor at generalizing. To address this, we evaluate three modifications of BiLSTM-CRF for BioNER to improve generalization: improved regularization via variational dropout, transfer learning, and multi-task learning.
RESULTS: We measure the effect that each strategy has when training/testing on the same corpus (‟in-corpus" performance) and when training on one corpus and evaluating on another (‟out-of-corpus" performance), our measure of the model's ability to generalize. We found that variational dropout improves out-of-corpus performance by an average of 4.62%, transfer learning by 6.48% and multi-task learning by 8.42%. The maximal increase we identified combines multi-task learning and variational dropout, which boosts out-of-corpus performance by 10.75%. Furthermore, we make available a new open-source tool, called Saber, that implements our best BioNER models.
AVAILABILITY: Source code for our biomedical IE tool is available at https://github.com/BaderLab/saber. Corpora and other resources used in this study are available at https://github.com/BaderLab/Towards-reliable-BioNER.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PMID: 31218364 [PubMed - as supplied by publisher]



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Evaluating the predictions of the protein stability change upon single amino acid substitutions for the FXN CAGI5 challenge.

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Evaluating the predictions of the protein stability change upon single amino acid substitutions for the FXN CAGI5 challenge.

Hum Mutat. 2019 Jun 17;:

Authors: Savojardo C, Petrosino M, Babbi G, Bovo S, Corbi-Verge C, Casadio R, Fariselli P, Folkman L, Garg A, Karimi M, Katsonis P, Kim PM, Lichtarge O, Martelli PL, Pasquo A, Pal D, Shen Y, Strokach AV, Turina P, Zhou Y, Andreoletti G, Brenner S, Chiaraluce R, Consalvi V, Capriotti E

Abstract
Frataxin (FXN) is a highly-conserved protein found in prokaryotes and eukaryotes that is required for an efficient regulation of cellular iron homeostasis. Experimental evidence associates amino acid substitutions of the frataxin to Friedreich Ataxia, a neurodegenerative disorder. Recently, new thermodynamic experiments have been performed to study the impact of somatic variations identified in cancer tissues on protein stability. The Critical Assessment of Genome Interpretation (CAGI) data provider at the University of Rome measured the unfolding free energy of a set of variants (frataxin challenge dataset) with far-UV circular dichroism and intrinsic fluorescence spectra. These values have been used to calculate the change in unfolding free energy between the variant and wild-type proteins at zero concentration of denaturant (ΔΔGH2O ). The frataxin challenge dataset, composed of eight amino acid substitutions, was used to evaluate the performance of the current computational methods for predicting the ΔΔGH2O value associated with the variants and to classify them as destabilizing and not destabilizing. For the fifth edition of CAGI, six independent research groups from Asia, Australia, Europe and North America submitted 12 sets of predictions from different approaches. In this paper we report the results of our assessment and discuss the limitations of the tested algorithms. This article is protected by copyright. All rights reserved.

PMID: 31209948 [PubMed - as supplied by publisher]



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Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells.

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Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells.

Stem Cell Res Ther. 2019 Jun 13;10(1):166

Authors: Ahlfors JE, Azimi A, El-Ayoubi R, Velumian A, Vonderwalde I, Boscher C, Mihai O, Mani S, Samoilova M, Khazaei M, Fehlings MG, Morshead CM

Abstract
BACKGROUND: Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window.
METHODS: We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry.
RESULTS: Human somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes.
CONCLUSIONS: Human drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

PMID: 31196173 [PubMed - in process]



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