Publications by authors named "Graham J Lieschke"

70 Publications

Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions.

Dis Model Mech 2021 Jul 23;14(7). Epub 2021 Jul 23.

Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia.

Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.047431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319549PMC
July 2021

Pioneer neutrophils release chromatin within in vivo swarms.

Elife 2021 07 21;10. Epub 2021 Jul 21.

The Bateson Centre and Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield Medical School, Sheffield, United Kingdom.

Neutrophils are rapidly recruited to inflammatory sites where their coordinated migration forms clusters, a process termed neutrophil swarming. The factors that modulate early stages of neutrophil swarming are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around an individual pioneer neutrophil. We observed the violent release of extracellular cytoplasmic and nuclear fragments by the pioneer and early swarming neutrophils. By combining in vitro and in vivo approaches to study essential components of neutrophil extracellular traps (NETs), we provide in-depth characterisation and high-resolution imaging of the composition and morphology of these release events. Using a photoconversion approach to track neutrophils within developing swarms, we identify that the fate of swarm-initiating pioneer neutrophils involves extracellular chromatin release and that the key NET components gasdermin, neutrophil elastase, and myeloperoxidase are required for the swarming process. Together our findings demonstrate that release of cellular components by pioneer neutrophils is an initial step in neutrophil swarming at sites of tissue injury.
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http://dx.doi.org/10.7554/eLife.68755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298094PMC
July 2021

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Nature 2021 03 10;591(7849):281-287. Epub 2021 Feb 10.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.
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http://dx.doi.org/10.1038/s41586-021-03199-7DOI Listing
March 2021

The Resistance to Host Antimicrobial Peptides in Infections Caused by Daptomycin-Resistant .

Antibiotics (Basel) 2021 Jan 20;10(2). Epub 2021 Jan 20.

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, VIC 3800, Australia.

Daptomycin is an important antibiotic for the treatment of infections caused by . The emergence of daptomycin resistance in is associated with treatment failure and persistent infections with poor clinical outcomes. Here, we investigated host innate immune responses against clinically derived, daptomycin-resistant (DAP-R) and -susceptible paired isolates using a zebrafish infection model. We showed that the control of DAP-R infections was attenuated in vivo due to cross-resistance to host cationic antimicrobial peptides. These data provide mechanistic understanding into persistent infections caused by DAP-R and provide crucial insights into the adaptive evolution of this troublesome pathogen.
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http://dx.doi.org/10.3390/antibiotics10020096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908987PMC
January 2021

Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy.

J Leukoc Biol 2020 08 23;108(2):455-468. Epub 2020 Apr 23.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

Neutrophil and macrophage (Mϕ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.
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http://dx.doi.org/10.1002/JLB.3HI0120-589RDOI Listing
August 2020

Utility of clinical comprehensive genomic characterization for diagnostic categorization in patients presenting with hypocellular bone marrow failure syndromes.

Haematologica 2021 01 1;106(1):64-73. Epub 2021 Jan 1.

Clinical Haematology, Peter MacCallum Cancer Centre/Royal Melbourne Hospital, Melbourne, Australia.

Bone marrow failure (BMF) related to hypoplasia of hematopoietic elements in the bone marrow is a heterogeneous clinical entity with a broad differential diagnosis including both inherited and acquired causes. Accurate diagnostic categorization is critical to optimal patient care and detection of genomic variants in these patients may provide this important diagnostic and prognostic information. We performed real-time, accredited (ISO15189) comprehensive genomic characterization including targeted sequencing and whole exome sequencing in 115 patients with BMF syndrome (median age 24 years, range 3 months - 81 years). In patients with clinical diagnoses of inherited BMF syndromes, acquired BMF syndromes or clinically unclassifiable BMF we detected variants in 52% (12/23), 53% (25/47) and 56% (25/45) respectively. Genomic characterization resulted in a change of diagnosis in 30/115 (26%) including the identification of germline causes for 3/47 and 16/45 cases with pre-test diagnoses of acquired and clinically unclassifiable BMF respectively. The observed clinical impact of accurate diagnostic categorization included choice to perform allogeneic stem cell transplantation, disease-specific targeted treatments, identification of at-risk family members and influence of sibling allogeneic stem cell donor choice. Multiple novel pathogenic variants and copy number changes were identified in our cohort including in TERT, FANCA, RPS7 and SAMD9. Whole exome sequence analysis facilitated the identification of variants in two genes not typically associated with a primary clinical manifestation of BMF but also demonstrated reduced sensitivity for detecting low level acquired variants. In conclusion, genomic characterization can improve diagnostic categorization of patients presenting with hypoplastic BMF syndromes and should be routinely performed in this group of patients.
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http://dx.doi.org/10.3324/haematol.2019.237693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776333PMC
January 2021

β-glucan-dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment.

PLoS Biol 2019 09 4;17(9):e3000113. Epub 2019 Sep 4.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called "shuttling." In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, and involves alterations of phagocyte mobility, intercellular tethering, and phagosome transfer. Shuttling kinetics were fungal-species-specific, implicating a fungal determinant. β-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favorable macrophage intracellular niche. Shuttling is a frequent host-pathogen interaction contributing to fungal infection establishment patterns.
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http://dx.doi.org/10.1371/journal.pbio.3000113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746390PMC
September 2019

Antibiotic resistance and host immune evasion in mediated by a metabolic adaptation.

Proc Natl Acad Sci U S A 2019 02 11;116(9):3722-3727. Epub 2019 Feb 11.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, VIC 3800, Australia;

is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, , encoding cardiolipin synthase. Single point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The point mutations also allowed to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.
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http://dx.doi.org/10.1073/pnas.1812066116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397524PMC
February 2019

Hematopoietic growth factors: the scenario in zebrafish.

Growth Factors 2018 12 15;36(5-6):196-212. Epub 2019 Feb 15.

a Australian Regenerative Medicine Institute, Monash University , Clayton , Australia.

Humoral regulation by ligand/receptor interactions is a fundamental feature of vertebrate hematopoiesis. Zebrafish are an established vertebrate animal model of hematopoiesis, sharing with mammals conserved genetic, molecular and cell biological regulatory mechanisms. This comprehensive review considers zebrafish hematopoiesis from the perspective of the hematopoietic growth factors (HGFs), their receptors and their actions. Zebrafish possess multiple HGFs: CSF1 (M-CSF) and CSF3 (G-CSF), kit ligand (KL, SCF), erythropoietin (EPO), thrombopoietin (THPO/TPO), and the interleukins IL6, IL11, and IL34. Some ligands and/or receptor components have been duplicated by various mechanisms including the teleost whole genome duplication, adding complexity to the ligand/receptor interactions possible, but also providing examples of several different outcomes of ligand and receptor subfunctionalization or neofunctionalization. CSF2 (GM-CSF), IL3 and IL5 and their receptors are absent from zebrafish. Overall the humoral regulation of hematopoiesis in zebrafish displays considerable similarity with mammals, which can be applied in biological and disease modelling research.
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http://dx.doi.org/10.1080/08977194.2019.1567506DOI Listing
December 2018

The Neutrophil Nucleus: An Important Influence on Neutrophil Migration and Function.

Front Immunol 2018 4;9:2867. Epub 2018 Dec 4.

Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia.

Neutrophil nuclear morphology has historically been used in haematology for neutrophil identification and characterisation, but its exact role in neutrophil function has remained enigmatic. During maturation, segmentation of the neutrophil nucleus into its mature, multi-lobulated shape is accompanied by distinct changes in nuclear envelope composition, resulting in a unique nucleus that is believed to be imbued with extraordinary nuclear flexibility. As a rate-limiting factor for cell migration, nuclear morphology and biomechanics are particularly important in the context of neutrophil migration during immune responses. Being an extremely plastic and fast migrating cell type, it is to be expected that neutrophils have an especially deformable nucleus. However, many questions still surround the dynamic capacities of the neutrophil nucleus, and which nuclear and cytoskeletal elements determine these dynamics. The biomechanics of the neutrophil nucleus should also be considered for their influences on the production of neutrophil extracellular traps (NETs), given this process sees the release of chromatin "nets" from nucleoplasm to extracellular space. Although past studies have investigated neutrophil nuclear composition and shape, in a new era of more sophisticated biomechanical and genetic techniques, 3D migration studies, and higher resolution microscopy we now have the ability to further investigate and understand neutrophil nuclear plasticity at an unprecedented level. This review addresses what is currently understood about neutrophil nuclear structure and its role in migration and the release of NETs, whilst highlighting open questions surrounding neutrophil nuclear dynamics.
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http://dx.doi.org/10.3389/fimmu.2018.02867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288403PMC
October 2019

Toxicological assessment of additively manufactured methacrylates for medical devices in dentistry.

Acta Biomater 2018 09 10;78:64-77. Epub 2018 Aug 10.

School of Dentistry and Oral Health, Griffith Health, Griffith University, Australia. Electronic address:

The paucity of information on the biological risks of photopolymers in additive manufacturing is a major challenge for the uptake of the technology in the construction of medical devices in dentistry. In this paper, the biocompatibility of methacrylates for denture bases, splints, retainers and surgical guides were evaluated using the innovative zebrafish embryo model, which is providing a high potential for toxicity profiling of photopolymers and has high genetic similarity to humans. Toxicological data obtained confirmed gradations of toxicity influenced by ethanol treatment, exposure scenarios and extraction vehicles. In direct exposure tests, juvenile fish exposed to non-treated methacrylates in ultrapure water showed accelerated toxicity endpoints compared to fish in transparent E3 medium. Similarly, toxic extracts induced mostly acute responses (embryonic mortality) in contrast to cumulative chronic (sublethal and teratogenic effects) in direct exposure. Methacrylates composed of >60% Ethoxylated bisphenol A dimethacrylate produced a relatively lower conversion rate in FTIR spectroscopy, but were safe in zebrafish bioassays after ethanol treatment. The study affirms that biocompatibility was influenced primarily by physico-chemical characteristics of the materials, which subsequently influenced their residual monomer content before and after immersion in ethanol. Given the precautionary implications of the study, we propose a 3-tiered approach i.e. using approved materials, apposite manufacturing parameters and post-processing techniques that together guarantee optimal results for medical devices.

Statement Of Significance: This study is timely and relevant since there is limited published literature that precisely describes the toxicological properties of additively manufactured methacrylates despite their increased popularity for medical devices. While it is generally accepted that the zebrafish excels as a model system for developmental toxicity, a further examination of its utility in this study using different protocols provides basis for its consideration and adoption at a crucial time when there is a lack of consensus regarding the most suited biological assessment methods for medical devices.
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http://dx.doi.org/10.1016/j.actbio.2018.08.007DOI Listing
September 2018

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.

PLoS Pathog 2018 06 8;14(6):e1007063. Epub 2018 Jun 8.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.
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http://dx.doi.org/10.1371/journal.ppat.1007063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010348PMC
June 2018

Blocking fatty acid-fueled mROS production within macrophages alleviates acute gouty inflammation.

J Clin Invest 2018 05 26;128(5):1752-1771. Epub 2018 Mar 26.

Department of Molecular Medicine and Pathology and.

Gout is the most common inflammatory arthritis affecting men. Acute gouty inflammation is triggered by monosodium urate (MSU) crystal deposition in and around joints that activates macrophages into a proinflammatory state, resulting in neutrophil recruitment. A complete understanding of how MSU crystals activate macrophages in vivo has been difficult because of limitations of live imaging this process in traditional animal models. By live imaging the macrophage and neutrophil response to MSU crystals within an intact host (larval zebrafish), we reveal that macrophage activation requires mitochondrial ROS (mROS) generated through fatty acid oxidation. This mitochondrial source of ROS contributes to NF-κB-driven production of IL-1β and TNF-α, which promote neutrophil recruitment. We demonstrate the therapeutic utility of this discovery by showing that this mechanism is conserved in human macrophages and, via pharmacologic blockade, that it contributes to neutrophil recruitment in a mouse model of acute gouty inflammation. To our knowledge, this study is the first to uncover an immunometabolic mechanism of macrophage activation that operates during acute gouty inflammation. Targeting this pathway holds promise in the management of gout and, potentially, other macrophage-driven diseases.
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http://dx.doi.org/10.1172/JCI94584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919807PMC
May 2018

Intron retention enhances gene regulatory complexity in vertebrates.

Genome Biol 2017 11 16;18(1):216. Epub 2017 Nov 16.

Gene & Stem Cell Therapy Program, Centenary Institute, University of Sydney, Camperdown, 2050, NSW, Australia.

Background: While intron retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution.

Results: Our RNA-sequencing-based analysis suggests that IR increases gene regulatory complexity, which is indicated by a strong anti-correlation between the number of genes affected by IR and the number of protein-coding genes in the genome of individual species. Our results confirm that IR affects many orthologous or functionally related genes in granulocytes. Further analysis uncovers new and unanticipated conserved characteristics of intron-retaining transcripts. We find that intron-retaining genes are transcriptionally co-regulated from bidirectional promoters. Intron-retaining genes have significantly longer 3' UTR sequences, with a corresponding increase in microRNA binding sites, some of which include highly conserved sequence motifs. This suggests that intron-retaining genes are highly regulated post-transcriptionally.

Conclusions: Our study provides unique insights concerning the role of IR as a robust and evolutionarily conserved mechanism of gene expression regulation. Our findings enhance our understanding of gene regulatory complexity by adding another contributor to evolutionary adaptation.
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http://dx.doi.org/10.1186/s13059-017-1339-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688624PMC
November 2017

Splicing dysfunction and disease: The case of granulopoiesis.

Semin Cell Dev Biol 2018 03 1;75:23-39. Epub 2017 Sep 1.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia. Electronic address:

Splicing is a ubiquitous process in eukaryotic cells, long recognised as contributing to diversity of the transcriptome. More specifically, splicing fine-tunes the transcriptome output for highly individual outcomes at different stages of cell development, in specific timeframes, which when perturbed result in significant human diseases. Granulopoiesis provides a particularly well studied example of how splicing can be a highly flexible but tightly regulated process. Focusing on the specific case of granulopoiesis, this review surveys the contribution of cis-splicing variations in individual genes and the trans-regulation of global splicing outcomes during the normal development of neutrophils. Further, the contribution of splicing dysfunction to the pathogenesis of diseases of neutrophil number, function and maturation including hereditary neutropenia, myelodysplasia, and acute myeloid leukaemia is explored.
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http://dx.doi.org/10.1016/j.semcdb.2017.08.048DOI Listing
March 2018

The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger.

Nat Commun 2017 04 6;8:14911. Epub 2017 Apr 6.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.

In response to infection and injury, the neutrophil population rapidly expands and then quickly re-establishes the basal state when inflammation resolves. The exact pathways governing neutrophil/macrophage lineage outputs from a common granulocyte-macrophage progenitor are still not completely understood. From a forward genetic screen in zebrafish, we identify the transcriptional repressor, ZBTB11, as critical for basal and emergency granulopoiesis. ZBTB11 sits in a pathway directly downstream of master myeloid regulators including PU.1, and TP53 is one direct ZBTB11 transcriptional target. TP53 repression is dependent on ZBTB11 cys116, which is a functionally critical, metal ion-coordinating residue within a novel viral integrase-like zinc finger domain. To our knowledge, this is the first description of a function for this domain in a cellular protein. We demonstrate that the PU.1-ZBTB11-TP53 pathway is conserved from fish to mammals. Finally, Zbtb11 mutant rescue experiments point to a ZBTB11-regulated TP53 requirement in development of other organs.
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http://dx.doi.org/10.1038/ncomms14911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384227PMC
April 2017

Chromatin-remodeling factor SMARCD2 regulates transcriptional networks controlling differentiation of neutrophil granulocytes.

Nat Genet 2017 May 3;49(5):742-752. Epub 2017 Apr 3.

Department of Pediatrics, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

We identify SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2), also known as BAF60b (BRG1/Brahma-associated factor 60b), as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells, and various developmental aberrations, we identified three homozygous loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPɛ and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and is a potential tumor suppressor in leukemia.
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http://dx.doi.org/10.1038/ng.3833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885283PMC
May 2017

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia.

Sci Rep 2017 03 10;7:44455. Epub 2017 Mar 10.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.

Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life.
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http://dx.doi.org/10.1038/srep44455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345067PMC
March 2017

MED12 in hematopoietic stem cells-cell specific function despite ubiquitous expression.

Stem Cell Investig 2017 18;4. Epub 2017 Jan 18.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.

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http://dx.doi.org/10.21037/sci.2016.12.04DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5313294PMC
January 2017

Acinetobacter baumannii phenylacetic acid metabolism influences infection outcome through a direct effect on neutrophil chemotaxis.

Proc Natl Acad Sci U S A 2016 08 9;113(34):9599-604. Epub 2016 Aug 9.

Infection and Immunity Program, Monash Biomedicine Discovery Institute, Melbourne, VIC 3800, Australia; Department of Microbiology, Monash University, Melbourne, VIC 3800, Australia; Department of Infectious Diseases, The Alfred Hospital and Central Clinical School, Monash University, Melbourne, VIC 3004, Australia

Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate disease.
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http://dx.doi.org/10.1073/pnas.1523116113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003227PMC
August 2016

A zebrafish model of inflammatory lymphangiogenesis.

Biol Open 2015 Sep 14;4(10):1270-80. Epub 2015 Sep 14.

Department of Molecular Medicine & Pathology, School of Medical Sciences, University of Auckland, Auckland 1142, New Zealand

Inflammatory bowel disease (IBD) is a disabling chronic inflammatory disease of the gastrointestinal tract. IBD patients have increased intestinal lymphatic vessel density and recent studies have shown that this may contribute to the resolution of IBD. However, the molecular mechanisms involved in IBD-associated lymphangiogenesis are still unclear. In this study, we established a novel inflammatory lymphangiogenesis model in zebrafish larvae involving colitogenic challenge stimulated by exposure to 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sodium sulphate (DSS). Treatment with either TNBS or DSS resulted in vascular endothelial growth factor receptor (Vegfr)-dependent lymphangiogenesis in the zebrafish intestine. Reduction of intestinal inflammation by the administration of the IBD therapeutic, 5-aminosalicylic acid, reduced intestinal lymphatic expansion. Zebrafish macrophages express vascular growth factors vegfaa, vegfc and vegfd and chemical ablation of these cells inhibits intestinal lymphatic expansion, suggesting that the recruitment of macrophages to the intestine upon colitogenic challenge is required for intestinal inflammatory lymphangiogenesis. Importantly, this study highlights the potential of zebrafish as an inflammatory lymphangiogenesis model that can be used to investigate the role and mechanism of lymphangiogenesis in inflammatory diseases such as IBD.
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http://dx.doi.org/10.1242/bio.013540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610225PMC
September 2015

Myeloid Growth Factors Promote Resistance to Mycobacterial Infection by Curtailing Granuloma Necrosis through Macrophage Replenishment.

Cell Host Microbe 2015 Jul;18(1):15-26

Department of Medicine, University of Cambridge, Cambridge CB2 0QH, UK; Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Immunology, University of Washington, Seattle, WA 98195, USA; Department of Medicine, University of Washington, Seattle, WA 98195, USA. Electronic address:

The mycobacterial ESX-1 virulence locus accelerates macrophage recruitment to the forming tuberculous granuloma. Newly recruited macrophages phagocytose previously infected apoptotic macrophages to become new bacterial growth niches. Granuloma macrophages can then necrose, releasing mycobacteria into the extracellular milieu, which potentiates their growth even further. Using zebrafish with genetic or pharmacologically induced macrophage deficiencies, we find that global macrophage deficits increase susceptibility to mycobacterial infection by accelerating granuloma necrosis. This is because reduction in the macrophage supply below a critical threshold decreases granuloma macrophage replenishment to the point where apoptotic infected macrophages, failing to get engulfed, necrose. Reducing macrophage demand by removing bacterial ESX-1 offsets the susceptibility of macrophage deficits. Conversely, increasing macrophage supply in wild-type fish by overexpressing myeloid growth factors induces resistance by curtailing necrosis. These findings may explain the susceptibility of humans with mononuclear cytopenias to mycobacterial infections and highlight the therapeutic potential of myeloid growth factors in tuberculosis.
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http://dx.doi.org/10.1016/j.chom.2015.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4509513PMC
July 2015

Delineating the roles of neutrophils and macrophages in zebrafish regeneration models.

Int J Biochem Cell Biol 2014 Nov 21;56:92-106. Epub 2014 Jul 21.

Australian Regenerative Medicine Institute, Monash University, Clayton 3800, Victoria, Australia. Electronic address:

The outcome following injury can be healing, scarring or regeneration, all of which initiate within a resolving inflammatory response. Regeneration, comprising the complete anatomical and functional restoration of lost tissue with minimal residual consequence of injury, is the outcome that most holistically restores prior function. Leukocytes are recognized as playing an important role in determining the balance between fully regenerative or only partially reparative outcomes. Although macrophages have attracted considerable attention for their capacity to direct pro-regenerative outcomes, neutrophils are also key players in initiating inflammation and in influencing its ensuing outcome. In the context of prior studies investigating the role of neutrophils and macrophages in wound healing and in tissue/organ regeneration (mostly wound repair/healing models in mice), we comprehensively review the experimental possibilities that zebrafish models offer for delineating the individual and interactive contributions of neutrophils and macrophages to the regenerative process in embryos and adults. Zebrafish are a highly regenerative vertebrate and have a myeloid system very analogous to that of less-regenerative mammalian models. There are well-characterized reporter lines for imaging and distinguishing neutrophil and macrophage behaviors in vivo, and tools enabling selective, independent manipulation of these two leukocyte lineages for functional studies. Zebrafish are an attractive model for delineating neutrophil and macrophage contributions not only to regeneration, but also to many other pathological processes. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.
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http://dx.doi.org/10.1016/j.biocel.2014.07.010DOI Listing
November 2014

Grainyhead-like 3 regulation of endothelin-1 in the pharyngeal endoderm is critical for growth and development of the craniofacial skeleton.

Mech Dev 2014 Aug 7;133:77-90. Epub 2014 Jun 7.

Department of Medicine, Monash University Central Clinical School, Prahran, VIC 3181, Australia; Alfred Hospital, Prahran, VIC 3181, Australia.

Craniofacial development is a highly conserved process that requires complex interactions between neural crest cells (NCCs) and pharyngeal tissues derived from all three germ layers. Signals emanating from the pharyngeal endoderm drive differentiation of NCCs into craniofacial cartilage, and disruption of this process underpins several human craniofacial defects (CFD). Here, we demonstrate that morpholino (MO)-mediated knockdown in zebrafish of the highly conserved transcription factor grainyhead-like 3 (grhl3), which is selectively expressed in the pharyngeal endoderm, leads to severe hypoplasia of the lower jaw cartilages. Phylogenetic analysis of conserved grhl-binding sites in gene regulatory regions identified endothelin-1 (edn1) as a putative direct grhl3 target gene, and this was confirmed by chromatin precipitation (ChIP) assays in zebrafish embryos. Injection of sub-phenotypic concentrations of MOs targeting both grhl3 and edn1 induced jaw abnormalities, and injection of edn1 mRNA into grhl3-morphants rescued both pharyngeal expression of the downstream effectors of edn1, and jaw cartilage formation. This study sheds new light on the role of endodermal endothelin-1 in vertebrate jaw development, and highlights potential new genetic defects that could underpin human CFD.
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http://dx.doi.org/10.1016/j.mod.2014.05.005DOI Listing
August 2014

Minor class splicing shapes the zebrafish transcriptome during development.

Proc Natl Acad Sci U S A 2014 Feb 10;111(8):3062-7. Epub 2014 Feb 10.

Ludwig Institute for Cancer Research, Melbourne-Parkville Branch, Parkville, VIC 3050, Australia.

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.
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http://dx.doi.org/10.1073/pnas.1305536111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939875PMC
February 2014

Real-time whole-body visualization of Chikungunya Virus infection and host interferon response in zebrafish.

PLoS Pathog 2013 5;9(9):e1003619. Epub 2013 Sep 5.

Institut Pasteur, Macrophages et Développement de l'Immunité, Department of Developmental and Stem Cells Biology, Paris, France ; CNRS URA2578, Paris, France ; Université Pierre et Marie Curie, Paris, France.

Chikungunya Virus (CHIKV), a re-emerging arbovirus that may cause severe disease, constitutes an important public health problem. Herein we describe a novel CHIKV infection model in zebrafish, where viral spread was live-imaged in the whole body up to cellular resolution. Infected cells emerged in various organs in one principal wave with a median appearance time of ∼14 hours post infection. Timing of infected cell death was organ dependent, leading to a shift of CHIKV localization towards the brain. As in mammals, CHIKV infection triggered a strong type-I interferon (IFN) response, critical for survival. IFN was mainly expressed by neutrophils and hepatocytes. Cell type specific ablation experiments further demonstrated that neutrophils play a crucial, unexpected role in CHIKV containment. Altogether, our results show that the zebrafish represents a novel valuable model to dynamically visualize replication, pathogenesis and host responses to a human virus.
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http://dx.doi.org/10.1371/journal.ppat.1003619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764224PMC
April 2014

PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model.

PLoS One 2013 30;8(8):e72636. Epub 2013 Aug 30.

MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield, United Kingdom.

Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB® m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072636PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758287PMC
April 2014

Immunoresponsive gene 1 augments bactericidal activity of macrophage-lineage cells by regulating β-oxidation-dependent mitochondrial ROS production.

Cell Metab 2013 Aug;18(2):265-78

Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, New Zealand.

Evidence suggests the bactericidal activity of mitochondria-derived reactive oxygen species (mROS) directly contributes to killing phagocytozed bacteria. Infection-responsive components that regulate this process remain incompletely understood. We describe a role for the mitochondria-localizing enzyme encoded by Immunoresponsive gene 1 (IRG1) during the utilization of fatty acids as a fuel for oxidative phosphorylation (OXPHOS) and associated mROS production. In a zebrafish infection model, infection-responsive expression of zebrafish irg1 is specific to macrophage-lineage cells and is regulated cooperatively by glucocorticoid and JAK/STAT signaling pathways. Irg1-depleted macrophage-lineage cells are impaired in their ability to utilize fatty acids as an energy substrate for OXPHOS-derived mROS production resulting in defective bactericidal activity. Additionally, the requirement for fatty acid β-oxidation during infection-responsive mROS production and bactericidal activity toward intracellular bacteria is conserved in murine macrophages. These results reveal IRG1 as a key component of the immunometabolism axis, connecting infection, cellular metabolism, and macrophage effector function.
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http://dx.doi.org/10.1016/j.cmet.2013.06.018DOI Listing
August 2013

In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation.

FEBS Lett 2013 Jul 25;587(14):2150-7. Epub 2013 May 25.

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia.

Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalophŏnus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre-mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal. Cph mutants accumulate aberrantly spliced transcripts retaining both U2- and U12-type introns. Within early haematopoiesis, myeloid differentiation is impaired, suggesting Prpf8 is required for haematopoietic development. Cph provides an animal model for zygotic PRPF8 dysfunction diseases and for evaluating therapeutic interventions.
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http://dx.doi.org/10.1016/j.febslet.2013.05.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820954PMC
July 2013

Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted ribosome biogenesis.

PLoS Genet 2013 7;9(2):e1003279. Epub 2013 Feb 7.

Colon Molecular and Cellular Biology Laboratory, Ludwig Institute for Cancer Research, Melbourne-Parkville Branch, Melbourne, Victoria, Australia.

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
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http://dx.doi.org/10.1371/journal.pgen.1003279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567153PMC
June 2013
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