Donnelly Centre for Cellular and Biomolecular Research

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Hypoxia-inducible factor drives vascularization of modularly assembled engineered tissue.

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Hypoxia-inducible factor drives vascularization of modularly assembled engineered tissue.

Tissue Eng Part A. 2018 Dec 26;:

Authors: Lam GC, Sefton MV

Abstract
Robust vascularization is critical for engineering tissues of clinically relevant size and cell loads. Delineating the rate limiting steps underlying vascularization is necessary to creating strategies for faster, better vascularization of tissue constructs. We used two inhibitory methods to dissect the role of hypoxia inducible factor (HIF) in vascularization-inducing engineered tissues, here constructed from self-assembly of sub-millimeter sized tissues injected subcutaneously. Both systemic pharmacological inhibition using digoxin, and genetic inhibition (shRNA-transduced endothelial cells) reduced vascularization and oxygenation within constructs, but elicited different mechanisms of action. Systemic inhibition negatively impacted early (day 3) recruitment of host-derived endothelial progenitor cells and macrophages/monocytes to the implant. Genetic inhibition in graft-derived endothelial cells impaired their survival in low serum conditions as well as their baseline angiogenic function. Together, our study demonstrates that HIF is an important driver of vascularization in tissue constructs. While hypoxia is assumed to be an important feature of tissue engineering, this study directly connects inhibition of vascularization to HIF inhibition.

PMID: 30585759 [PubMed - as supplied by publisher]



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Influencing neuroplasticity in stroke treatment with advanced biomaterials-based approaches.

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Influencing neuroplasticity in stroke treatment with advanced biomaterials-based approaches.

Adv Drug Deliv Rev. 2018 Dec 20;:

Authors: Obermeyer JM, Gracias A, Ho E, Shoichet MS

Abstract
Since the early 1990s, we have known that the adult brain is not static and has the capacity to repair itself. The delivery of various therapeutic factors and cells have resulted in some exciting pre-clinical and clinical outcomes in stroke models by targeting post-injury plasticity to enhance recovery. Developing a deeper understanding of the pathways that modulate plasticity will enable us to optimize delivery strategies for therapeutics and achieve more robust effects. Biomaterials are a key tool for the optimization of these potential treatments, owing to their biocompatibility and tunability. In this review, we identify factors and targets that impact plastic processes known to contribute to recovery, discuss the role of biomaterials in enhancing the efficacy of treatment strategies, and suggest combinatorial approaches based on the stage of injury progression.

PMID: 30579882 [PubMed - as supplied by publisher]



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The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans.

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The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans.

Elife. 2018 12 21;7:

Authors: Davis GM, Tu S, Anderson JW, Colson RN, Gunzburg MJ, Francisco MA, Ray D, Shrubsole SP, Sobotka JA, Seroussi U, Lao RX, Maity T, Wu MZ, McJunkin K, Morris QD, Hughes TR, Wilce JA, Claycomb JM, Weng Z, Boag PR

Abstract
Proper regulation of germline gene expression is essential for fertility and maintaining species integrity. In the C. elegans germline, a diverse repertoire of regulatory pathways promote the expression of endogenous germline genes and limit the expression of deleterious transcripts to maintain genome homeostasis. Here we show that the conserved TRIM-NHL protein, NHL-2, plays an essential role in the C. elegans germline, modulating germline chromatin and meiotic chromosome organization. We uncover a role for NHL-2 as a co-factor in both positively (CSR-1) and negatively (HRDE-1) acting germline 22G-small RNA pathways and the somatic nuclear RNAi pathway. Furthermore, we demonstrate that NHL-2 is a bona fide RNA binding protein and, along with RNA-seq data point to a small RNA independent role for NHL-2 in regulating transcripts at the level of RNA stability. Collectively, our data implicate NHL-2 as an essential hub of gene regulatory activity in both the germline and soma.

PMID: 30575518 [PubMed - indexed for MEDLINE]



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Identifying Pseudomonas syringae Type III Secreted Effector Function via a Yeast Genomic Screen.

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Identifying Pseudomonas syringae Type III Secreted Effector Function via a Yeast Genomic Screen.

G3 (Bethesda). 2018 Dec 20;:

Authors: Huei-Yi Lee A, Bastedo DP, Youn JY, Lo T, Middleton MA, Kireeva I, Lee JY, Sharifpoor S, Baryshnikova A, Zhang J, Wang PW, Peisajovich SG, Costanzo M, Andrews BJ, Boone CM, Desveaux D, Guttman DS

Abstract
Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae. This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to identify potential host targets of P. syringae T3SEs. We focused on the acetyltransferase, HopZ1a, which interacts with plant tubulin and alters microtubule networks. To uncover putative HopZ1a host targets, we identified yeast genes with genetic interaction profiles most similar (i.e. congruent) to the PGA profile of HopZ1a and performed a functional enrichment analysis of these HopZ1a-congruent genes. We compared the congruence analyses above to previously described HopZ physical interaction datasets and identified kinesins as potential HopZ1a targets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1, illustrating the utility of our T3SE-expressing yeast library to characterize T3SE functions.

PMID: 30573466 [PubMed - as supplied by publisher]



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Biosynthetic Oligoclonal Antivenom (BOA) for Snakebite and Next-Generation Treatments for Snakebite Victims.

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Biosynthetic Oligoclonal Antivenom (BOA) for Snakebite and Next-Generation Treatments for Snakebite Victims.

Toxins (Basel). 2018 Dec 13;10(12):

Authors: Kini RM, Sidhu SS, Laustsen AH

Abstract
Snakebite envenoming is a neglected tropical disease that each year claims the lives of 80,000⁻140,000 victims worldwide. The only effective treatment against envenoming involves intravenous administration of antivenoms that comprise antibodies that have been isolated from the plasma of immunized animals, typically horses. The drawbacks of such conventional horse-derived antivenoms include their propensity for causing allergenic adverse reactions due to their heterologous and foreign nature, an inability to effectively neutralize toxins in distal tissue, a low content of toxin-neutralizing antibodies, and a complex manufacturing process that is dependent on husbandry and procurement of snake venoms. In recent years, an opportunity to develop a fundamentally novel type of antivenom has presented itself. By using modern antibody discovery strategies, such as phage display selection, and repurposing small molecule enzyme inhibitors, next-generation antivenoms that obviate the drawbacks of existing plasma-derived antivenoms could be developed. This article describes the conceptualization of a novel therapeutic development strategy for biosynthetic oligoclonal antivenom (BOA) for snakebites based on recombinantly expressed oligoclonal mixtures of human monoclonal antibodies, possibly combined with repurposed small molecule enzyme inhibitors.

PMID: 30551565 [PubMed - in process]



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Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response.

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Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response.

J Biol Chem. 2019 01 25;294(4):1396-1409

Authors: Turowec JP, Lau EWT, Wang X, Brown KR, Fellouse FA, Jawanda KK, Pan J, Moffat J, Sidhu SS

Abstract
Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approved cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined antibody engineering and functional genomics screens to identify putative mechanisms of resistance or synthetic lethality with antibody-mediated anti-proliferative effects. We developed a synthetic antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization, and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 antibody therapeutics.

PMID: 30523157 [PubMed - indexed for MEDLINE]



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Rapid and Accurate Structure-Based Therapeutic Peptide Design using GPU Accelerated Thermodynamic Integration.

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Rapid and Accurate Structure-Based Therapeutic Peptide Design using GPU Accelerated Thermodynamic Integration.

Proteins. 2018 Dec 06;:

Authors: Garton M, Corbi-Verge C, Hu Y, Nim S, Tarasova N, Sherborne B, Kim PM

Abstract
Peptide-based therapeutics are an alternative to small molecule drugs as they offer superior specificity, lower toxicity and easy synthesis. Here we present an approach that leverages the dramatic performance increase afforded by the recent arrival of GPU accelerated thermodynamic integration (TI). GPU TI facilitates very fast, highly accurate binding affinity optimization of peptides against therapeutic targets. We benchmarked TI predictions using published peptide binding optimization studies. Prediction of mutations involving charged side-chains was found to be less accurate than for non-charged, and use of a more complex 3-step TI protocol was found to boost accuracy in these cases. Using the 3-step protocol for non-charged side-chains either had no effect or was detrimental. We use the benchmarked pipeline to optimize a peptide binding to our recently discovered cancer target: EME1. TI calculations predict beneficial mutations using both canonical and non-canonical amino acids. We validate these predictions using fluorescence polarization and confirm that binding affinity is increased. We further demonstrate that this increase translates to a significant reduction in pancreatic cancer cell viability. This article is protected by copyright. All rights reserved.

PMID: 30520126 [PubMed - as supplied by publisher]



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A chemical defence against phage infection.

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A chemical defence against phage infection.

Nature. 2018 Dec 05;:

Authors: Kronheim S, Daniel-Ivad M, Duan Z, Hwang S, Wong AI, Mantel I, Nodwell JR, Maxwell KL

Abstract
The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.

PMID: 30518855 [PubMed - as supplied by publisher]



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Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.

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Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.

MBio. 2018 12 04;9(6):

Authors: Lee J, Mir A, Edraki A, Garcia B, Amrani N, Lou HE, Gainetdinov I, Pawluk A, Ibraheim R, Gao XD, Liu P, Davidson AR, Maxwell KL, Sontheimer EJ

Abstract
In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.

PMID: 30514786 [PubMed - indexed for MEDLINE]



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Mapping a diversity of genetic interactions in yeast.

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Mapping a diversity of genetic interactions in yeast.

Curr Opin Syst Biol. 2017 Dec;6:14-21

Authors: van Leeuwen J, Boone C, Andrews BJ

Abstract
Genetic interactions occur when the combination of multiple mutations yields an unexpected phenotype, and they may confound our ability to fully understand the genetic mechanisms underlying complex diseases. Genetic interactions are challenging to study because there are millions of possible different variant combinations within a given genome. Consequently, they have primarily been systematically explored in unicellular model organisms, such as yeast, with a focus on pairwise genetic interactions between loss-of-function alleles. However, there are many different types of genetic interactions, such as those occurring between gain-of-function or heterozygous mutations. Here, we review recent advances made in the systematic analysis of such diverse genetic interactions in yeast, and briefly discuss how similar studies could be undertaken in human cells.

PMID: 30505984 [PubMed]



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