Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

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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Neuro-genetic plasticity of Caenorhabditis elegans behavioral thermal tolerance.

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Neuro-genetic plasticity of Caenorhabditis elegans behavioral thermal tolerance.

BMC Neurosci. 2019 Jun 10;20(1):26

Authors: Stegeman GW, Medina D, Cutter AD, Ryu WS

Abstract
BACKGROUND: Animal responses to thermal stimuli involve intricate contributions of genetics, neurobiology and physiology, with temperature variation providing a pervasive environmental factor for natural selection. Thermal behavior thus exemplifies a dynamic trait that requires non-trivial phenotypic summaries to appropriately capture the trait in response to a changing environment. To characterize the deterministic and plastic components of thermal responses, we developed a novel micro-droplet assay of nematode behavior that permits information-dense summaries of dynamic behavioral phenotypes as reaction norms in response to increasing temperature (thermal tolerance curves, TTC).
RESULTS: We found that C. elegans TTCs shift predictably with rearing conditions and developmental stage, with significant differences between distinct wildtype genetic backgrounds. Moreover, after screening TTCs for 58 C. elegans genetic mutant strains, we determined that genes affecting thermosensation, including cmk-1 and tax-4, potentially play important roles in the behavioral control of locomotion at high temperature, implicating neural decision-making in TTC shape rather than just generalized physiological limits. However, expression of the transient receptor potential ion channel TRPA-1 in the nervous system is not sufficient to rescue rearing-dependent plasticity in TTCs conferred by normal expression of this gene, indicating instead a role for intestinal signaling involving TRPA-1 in the adaptive plasticity of thermal performance.
CONCLUSIONS: These results implicate nervous system and non-nervous system contributions to behavior, in addition to basic cellular physiology, as key mediators of evolutionary responses to selection from temperature variation in nature.

PMID: 31182018 [PubMed - in process]



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Local delivery of FK506 to injured peripheral nerve enhances axon regeneration after surgical nerve repair in rats.

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Local delivery of FK506 to injured peripheral nerve enhances axon regeneration after surgical nerve repair in rats.

Acta Biomater. 2019 May 26;:

Authors: Tajdaran K, Chan K, Shoichet MS, Gordon T, Borschel GH

Abstract
Administration of FK506, an FDA approved immunosuppressant, has been shown to enhance nerve regeneration following peripheral nerve injuries. However, the severe side effects of the systemically delivered FK506 has prevented clinicians from the routine use of the drug. In this study, we analyzed the effectiveness of our fibrin gel-based FK506 delivery system to promote axon regeneration in a rat peripheral nerve transection and immediate surgical repair model. In addition, biodistribution of FK506 from the local delivery system to the surrounding tissues was analyzed in vivo. Rats in the negative control groups either did not receive any delivery system treatment or received fibrin gel with empty microspheres. The experimental groups included rats treated with fibrin gel loaded with solubilized, particulate, and poly(lactic-co-glycolic) acid microspheres-encapsulated FK506. Rats in experimental groups receiving FK506 microspheres and the particulate FK506 regenerated the highest number of motor and sensory neurons. Histomorphometric analysis also demonstrated greater numbers of myelinated axons following particulate FK506 and FK506 microspheres treatment compared to the negative control groups. In biodistribution studies, FK506 was found at the nerve repair site, the sciatic nerve, and spinal cord, with little to no drug detection in other vital organs. Hence, the local application of FK506 via our delivery systems enhanced axon regeneration whilst avoiding the toxicity of systemic FK506. This local delivery strategy represents a new opportunity for clinicians to use for cases of peripheral nerve injuries. STATEMENT OF SIGNIFICANCE: This work for the first time investigated the influence of locally administered FK506 to the site of nerve injury and immediate repair directly on the number of motor and sensory neurons that regenerated their axons. Furthermore, using the immediate nerve repair model, we obtained valuable information about the biodistribution of FK506 within the nervous system following its release from the delivery system implanted at the site of nerve injury and repair. The strategy of local FK506 delivery holds a great promise in the clinical translation, as the localized delivery circumvents the main limitation of the systemic delivery of FK506, that of immunosuppression and toxicity.

PMID: 31141732 [PubMed - as supplied by publisher]



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Integrating yeast chemical genomics and mammalian cell pathway analysis.

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Integrating yeast chemical genomics and mammalian cell pathway analysis.

Acta Pharmacol Sin. 2019 May 28;:

Authors: Zhou FL, Li SC, Zhu Y, Guo WJ, Shao LJ, Nelson J, Simpkins S, Yang DH, Liu Q, Yashiroda Y, Xu JB, Fan YY, Yue JM, Yoshida M, Xia T, Myers CL, Boone C, Wang MW

Abstract
Chemical genomics has been applied extensively to evaluate small molecules that modulate biological processes in Saccharomyces cerevisiae. Here, we use yeast as a surrogate system for studying compounds that are active against metazoan targets. Large-scale chemical-genetic profiling of thousands of synthetic and natural compounds from the Chinese National Compound Library identified those with high-confidence bioprocess target predictions. To discover compounds that have the potential to function like therapeutic agents with known targets, we also analyzed a reference library of approved drugs. Previously uncharacterized compounds with chemical-genetic profiles resembling existing drugs that modulate autophagy and Wnt/β-catenin signal transduction were further examined in mammalian cells, and new modulators with specific modes of action were validated. This analysis exploits yeast as a general platform for predicting compound bioactivity in mammalian cells.

PMID: 31138898 [PubMed - as supplied by publisher]



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