ORCID Profile
0000-0002-8675-9426
Current Organisation
Centenary Institute
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Animal Behaviour | Life Histories | Genomics | Molecular Evolution | Ecology | Behavioural Ecology | Systems Biology | Genetics
Publisher: Springer Science and Business Media LLC
Date: 02-12-2012
Publisher: Research Square Platform LLC
Date: 06-01-2022
DOI: 10.21203/RS.3.RS-1172665/V1
Abstract: Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host factors controlling SARS-CoV-2 Spike binding. The top hit was a Toll-like receptor-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where it forms a cell surface complex with LRRC15 but does not support infection. Instead, LRRC15 functioned as a negative receptor suppressing both pseudotyped and live SARS-CoV-2 infection. LRRC15 is expressed in collagen-producing lung myofibroblasts where it can sequester virus and reduce infection in trans. Mechanistically LRRC15 is regulated by TGF-β, where moderate LRRC15 expression drives collagen production but high levels suppress it, revealing a novel lung fibrosis feedback circuit. Overall, LRRC15 is a master regulator of SARS-CoV-2, suppressing infection and controlling collagen production associated with “long-haul” COVID-19.
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.DEVCEL.2017.11.010
Abstract: The attenuation of ancestral pro-regenerative pathways may explain why humans do not efficiently regenerate damaged organs. Vertebrate lineages that exhibit robust regeneration, including the teleost zebrafish, provide insights into the maintenance of adult regenerative capacity. Using established models of spinal cord, heart, and retina regeneration, we discovered that zebrafish T
Publisher: Elsevier BV
Date: 07-2022
DOI: 10.1016/J.ENVPOL.2022.119081
Abstract: Per- and polyfluoroalkyl substances (PFAS) are ubiquitous in the environment and often ingested with food. PFAS exposure in people can have detrimental health consequences. Therefore, reducing PFAS burdens in food items is of great importance to public health. Here, we investigated whether cooking reduces PFAS concentrations in animal-derived food products by synthesizing experimental studies. Further, we examined the moderating effects of the following five variables: cooking time, liquid/animal tissue ratio, cooking temperature, carbon chain length of PFAS and the cooking category (oil-based, water-based & no-liquid cooking). In our systematic review searches, we obtained 512 effect sizes (relative differences in PFAS concentration between raw and cooked s les) from 10 relevant studies. These studies exclusively explored changes in PFAS concentrations in cooked seafood and freshwater fish. Our multilevel-meta-analysis has revealed that, on average, cooking reduced PFAS concentrations by 29%, although heterogeneity among effect sizes was very high (I
Publisher: American Diabetes Association
Date: 11-07-2017
DOI: 10.2337/DB16-1476
Abstract: Thyroid hormone (TH) signaling promotes tissue maturation and adult organ formation. Developmental transitions alter an organism's metabolic requirements, and it remains unclear how development and metabolic demands are coordinated. We used the zebrafish as a model to test whether and how TH signaling affects pancreatic islet maturation, and consequently glucose homeostasis, during the larval to juvenile transition. We found that exogenous TH precociously activates the β-cell differentiation genes pax6b and mnx1 while downregulating arxa, a master regulator of α-cell development and function. Together, these effects induced hypoglycemia, at least in part by increasing insulin and decreasing glucagon expression. We visualized TH target tissues using a novel TH-responsive reporter line and found that both α- and β-cells become targets of endogenous TH signaling during the larval-to-juvenile transition. Importantly, endogenous TH is required during this transition for the functional maturation of α- and β-cells in order to maintain glucose homeostasis. Thus, our study sheds new light on the regulation of glucose metabolism during major developmental transitions.
Publisher: Wiley
Date: 23-08-2019
DOI: 10.1111/MMI.14362
Publisher: Public Library of Science (PLoS)
Date: 25-07-2013
Publisher: Cold Spring Harbor Laboratory
Date: 18-12-2020
DOI: 10.1101/2020.12.17.423365
Abstract: DNA methylation predominantly occurs at CG dinucleotides in vertebrate genomes however, non-CG methylation (mCH) is also detectable in vertebrate tissues, most notably in the nervous system. In mammals it is well established that mCH is targeted to CAC trinucleotides by DNMT3A during nervous system development where it is enriched in gene bodies and associated with transcriptional repression. However, the conservation of developmental mCH accumulation and its deposition by DNMT3A is largely unexplored and has yet to be functionally demonstrated in other vertebrates. In this study, by analyzing DNA methylomes and transcriptomes of zebrafish brains, we identified enrichment of mCH at CAC trinucleotides (mCAC) at defined transposon motifs as well as in developmentally downregulated genes associated with developmental and neural functions. We further generated and analyzed DNA methylomes and transcriptomes of developing zebrafish larvae and demonstrated that, like in mammals, mCH accumulates during post-embryonic brain development. Finally, by employing CRISPR/Cas9 technology, we unraveled a conserved role for Dnmt3a enzymes in developmental mCAC deposition. Overall, this work demonstrates the evolutionary conservation of developmental mCH dynamics and highlights the potential of zebrafish as a model to study mCH regulation and function during normal and perturbed development.
Publisher: The Company of Biologists
Date: 18-02-2016
DOI: 10.1242/BIO.016295
Abstract: Here we genetically characterise pelvic finless, a naturally occurring model of hindlimb loss in zebrafish that lacks pelvic fin structures, which are homologous to tetrapod hindlimbs, but displays no other abnormalities. Using a hybrid positional cloning and next generation sequencing approach, we identified mutations in the nuclear localisation signal (NLS) of T-box transcription factor 4 (Tbx4) that impair nuclear localisation of the protein, resulting in altered gene expression patterns during pelvic fin development and the failure of pelvic fin development. Using a TALEN-induced tbx4 knockout allele we confirm that mutations within the Tbx4 NLS (A78V G79A) are sufficient to disrupt pelvic fin development. By combining histological, genetic, and cellular approaches we show that the hindlimb initiation gene tbx4 has an evolutionarily conserved, essential role in pelvic fin development. In addition, our novel viable model of hindlimb deficiency is likely to facilitate the elucidation of the detailed molecular mechanisms through which Tbx4 functions during pelvic fin and hindlimb development.
Publisher: Proceedings of the National Academy of Sciences
Date: 09-2009
Abstract: Pancreatic β-cells are critical regulators of glucose homeostasis, and they vary dramatically in their glucose stimulated metabolic response and levels of insulin secretion. It is unclear whether these parameters are influenced by the developmental origin of in idual β-cells. Using HOTcre, a Cre-based genetic switch that uses heat-induction to precisely control the temporal expression of transgenes, we labeled two populations of β-cells within the developing zebrafish pancreas. These populations originate in distinct pancreatic buds and exhibit gene expression profiles suggesting distinct functions during development. We find that the dorsal bud derived β-cells are quiescent and exhibit a marked decrease in insulin expression postembryonically. In contrast, ventral bud derived β-cells proliferate actively, and maintain high levels of insulin expression compared with dorsal bud derived β-cells. Therapeutic strategies to regulate β-cell proliferation and function are required to cure pathological states that result from excessive β-cell proliferation (e.g., insulinoma) or insufficient β-cell mass (e.g., diabetes mellitus). Our data reveal the existence of distinct populations of β-cells in vivo and should help develop better strategies to regulate β-cell differentiation and proliferation.
Publisher: Elsevier BV
Date: 10-2009
Publisher: Elsevier BV
Date: 11-2020
Publisher: Springer Science and Business Media LLC
Date: 23-05-2019
DOI: 10.1038/S42003-019-0444-0
Abstract: Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.
Publisher: Cold Spring Harbor Laboratory
Date: 07-06-2022
DOI: 10.1101/2022.06.07.495067
Abstract: Directed evolution uses cycles of gene ersification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or “VEGAS”) has been described. Here we report that the VEGAS system has major limitations precluding its use for directed evolution. The primary technical issue with the VEGAS system is an immediate contamination with “cheater” particles that bypass directed evolution circuits. By sequencing we find these cheater particles contain Sindbis structural genes instead of the intended directed evolution target transgene. These cheaters outcompete the VEGAS transgenes within 2 rounds of transduction but cannot themselves activate synthetic circuits that drive expression of Sindbis structural genes, preventing directed evolution c aigns. Similar results have been obtained in independent labs. Taken together, the VEGAS system does not work as described and, without significant redesign to suppress cheaters, cannot be used for mammalian directed evolution c aigns.
Publisher: Cold Spring Harbor Laboratory
Date: 10-11-2021
DOI: 10.1101/2021.11.09.467981
Abstract: Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here we use whole genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR -related cell surface receptor called leucine-rich repeat-containing protein 15 ( LRRC15 ). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe COVID-19 infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans . Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.
Publisher: Cold Spring Harbor Laboratory
Date: 30-12-2020
DOI: 10.1101/2020.12.30.424641
Abstract: COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use it to examine five public single-cell RNA-seq datasets with a total of 85 in idual s les. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate erse communication patterns across in iduals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients.
Publisher: Wiley
Date: 10-2021
Abstract: Per‐ and polyfluoroalkyl substances (PFAS) are a large group of manufactured chemicals. Since the beginning of their commercial production in the 1950s, PFAS have not only found their way into numerous industrial and commercial applications, but also into the bloodstream of much of the human population, the natural environment and wildlife. Exposure to high levels of PFAS poses a health risk for humans and animals, and may exacerbate the effects of other anthropogenic impacts faced by wildlife species. To gain a comprehensive overview of the abundance and distribution of PFAS research on wildlife species, and to better understand the drivers of this research, we will collate the available literature into a systematic evidence map and perform bibliometric analyses. The systematic mapping will present the distribution of research evidence that exists on PFAS in wildlife. The bibliometric analysis will provide an insight into the historical trends, interdisciplinarity, connectedness and the impact of the in idual papers. We will conduct a systematic literature search on Scopus, Web of Science and 10 other databases using predefined search strings. We will screen title, abstract and keywords first. We will then screen full‐text papers. Two reviewers will be involved in the screening process. We will only consider publications in English, peer‐reviewed articles, preprints and theses. We will include papers reporting concentrations of any of 34 main PFAS types (based on a previous study) in wild animals. We will assess all publications included in the systematic map for predetermined indicators of quality and potential study‐level biases. In addition, we will use bibliometric records from Scopus to perform impact and network analyses. We will present the results using a narrative summary, tables and colour‐coded maps, bar and network plots. Results and associated database will be available on a dedicated freely accessible website. This study will provide critical insight into the gaps and clusters of the literature with regard to the PFAS concentrations in wildlife. Our study will inform and direct future research efforts to fill the gaps revealed.
Publisher: eLife Sciences Publications, Ltd
Date: 13-12-2016
DOI: 10.7554/ELIFE.23065
Abstract: Certain microbes in the intestine secrete protein that stimulates the proliferation of beta cells in the pancreas during development.
Publisher: Elsevier BV
Date: 11-2004
DOI: 10.1016/J.CUB.2004.11.006
Abstract: Most developing organs are surrounded by an extracellular matrix (ECM), which must be remodeled to accommodate growth and morphogenesis. In C. elegans, the GON-1 ADAMTS metalloprotease regulates both elongation and shape of the developing gonad . Here, we report that either human ADAMTS-4 or ADAMTS-9 can substitute for GON-1 in transgenic worms, suggesting functional conservation between human and nematode homologs. We further identify fibulin (FBL-1), a widely conserved ECM component , as critical for gonadal morphogenesis. FBL-1 is expressed in nongonadal tissues but is present at the surface of the elongating gonad. A fibulin deletion mutant has a wider than normal gonad as well as body size defects. We find that GON-1 and fibulin have antagonistic roles in controlling gonadal shape. Depletion of fbl-1, but not other ECM components, rescues gon-1 elongation defects, and removal of gon-1 rescues fbl-1 width defects. Therefore, the GON-1 protease normally promotes tissue elongation and expansion, whereas the fibulin ECM protein blocks these key morphogenetic processes. We suggest that control of organ shape by GON-1 and fibulin in C. elegans may provide a model for similar cellular processes, including vasculogenesis, in humans.
Publisher: Elsevier BV
Date: 05-2021
Publisher: MDPI AG
Date: 09-08-2021
DOI: 10.3390/IJMS22168571
Abstract: Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.
Publisher: American Society for Microbiology
Date: 15-04-2018
DOI: 10.1128/JB.00654-17
Abstract: Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction of speB gene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448Δ pepO mutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections. IMPORTANCE Despite the continuing susceptibility of S. pyogenes to penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that control speB gene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator of speB gene expression in the globally disseminated M1T1 clone and as being essential for virulence.
Publisher: American Diabetes Association
Date: 16-08-2018
DOI: 10.2337/DB17-1223
Abstract: β-Cell loss and dysfunction play a critical role in the progression of type 1 and type 2 diabetes. Identifying new molecules and/or molecular pathways that improve β-cell function and/or increase β-cell mass should significantly contribute to the development of new therapies for diabetes. Using the zebrafish model, we screened 4,640 small molecules to identify modulators of β-cell function. This in vivo strategy identified 84 stimulators of insulin expression, which simultaneously reduced glucose levels. The insulin promoter activation kinetics for 32 of these stimulators were consistent with a direct mode of action. A subset of insulin stimulators, including the antidiabetic drug pioglitazone, induced the coordinated upregulation of gluconeogenic pck1 expression, suggesting functional response to increased insulin action in peripheral tissues. Notably, Kv1.3 inhibitors increased β-cell mass in larval zebrafish and stimulated β-cell function in adult zebrafish and in the streptozotocin-induced hyperglycemic mouse model. In addition, our data indicate that cytoplasmic Kv1.3 regulates β-cell function. Thus, using whole-organism screening, we have identified new small-molecule modulators of β-cell function and glucose metabolism.
Publisher: PeerJ
Date: 29-06-2018
DOI: 10.7717/PEERJ.5146
Abstract: Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during lification. Simultaneous exposure of single-stranded fragment ends, utilising the 3′→5′ exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.
Publisher: Elsevier BV
Date: 02-2018
DOI: 10.1016/J.DIABRES.2018.12.002
Abstract: Analyze cosegregation of aniridia and diabetes to identify genetic criteria for detection and early treatment of diabetes-susceptible aniridia patients. We assessed a two-generation family: three in iduals with aniridia, two previously diagnosed as type 2 diabetes. One in idual with aniridia, with unknown diabetes status, was evaluated by oral glucose tolerance test. Genetic analysis of aniridia-associated genes was performed on all available family members. Candidate genes were functionally tested by gene silencing in MIN6 pancreatic β-cells. A 25 year old male with aniridia had a diabetic oral glucose tolerance test despite a normal fasting blood glucose. A 484-630 kb deletion ∼120 kb distal to PAIRED BOX 6 (PAX6) showed dominant cosegregation with aniridia and diabetes in all affected family members. The deleted region contains regulatory elements for PAX6 expression and four additional coding regions. Knockdown of two of the deleted genes (Dnajc24 or Immp1l) with Pax6 impaired glucose-stimulated insulin secretion. We demonstrate dominant cosegregation of diabetes and aniridia with a deletion distal to PAX6, which is clinically distinct from the mild glucose intolerance previously reported with PAX6 coding mutations. Asymptomatic aniridia in iduals appear at risk of diabetes (and its complications) and could benefit from earlier diagnosis and treatment.
Publisher: Wiley
Date: 10-2022
DOI: 10.1002/ECE3.9423
Abstract: The obesity epidemic is concerning as obesity appears to negatively impact cognition and behavior. Furthermore, some studies suggest that this negative effect could be carried across generations from both mothers and fathers although evidence is not consistent. Here, we attempt to address how obesogenic diets in the parental generation (F0) can impact offspring's cognition and anxiety intergenerationally (F1) in a zebrafish model. We compare both mean trait values and their variances. Using a multifactorial design, we created a total of four groups: F1T (treatment mothers × treatment fathers) F1M (treatment mothers × control fathers) F1P (treatment fathers × control mothers) and F1C (control mothers × control fathers, F1C) and subjected them to anxiety tank tests and aversive learning assays. When both parents were exposed, offspring (F1T) displayed the poorest aversive learning, while offspring that only had one parent exposed (F1P and F1M) learnt the aversive learning task the best. Zebrafish in all groups displayed no statistically significant differences in anxiety‐associated behaviors. Males and females also performed similarly in both anxiety and aversive learning assays. While all F1 groups had similar levels of fasting blood glucose, variance in glucose levels were reduced in F1P and F1T indicating the importance of investigating heteroskedasticity between groups. Furthermore, anxiety behaviors of these two groups appeared to be less repeatable. To our knowledge, this is the first study to test the intergenerational effects of an obesogenic diet on zebrafish cognition. Our multifactorial design as well as repeated tests also allowed us to disentangle maternal and paternal effects (as well as combined effects) and accurately detect subtle information such as between‐in idual variation.
Publisher: American Chemical Society (ACS)
Date: 23-11-2018
Publisher: Springer Science and Business Media LLC
Date: 05-11-2020
DOI: 10.1038/S41598-020-75917-6
Abstract: Germline loss-of-function variation in TNFAIP3 , encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish ( Danio rerio ) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.
Publisher: PeerJ
Date: 17-01-2018
DOI: 10.7717/PEERJ.4292
Abstract: Zebrafish are increasingly used as a vertebrate model organism for various traits including swimming performance, obesity and metabolism, necessitating high-throughput protocols to generate standardized phenotypic information. Here, we propose a novel and cost-effective method for exercising zebrafish, using a coffee plunger and magnetic stirrer. To demonstrate the use of this method, we conducted a pilot experiment to show that this simple system provides repeatable estimates of maximal swim performance (intra-class correlation [ICC] = 0.34–0.41) and observe that exercise training of zebrafish on this system significantly increases their maximum swimming speed. We propose this high-throughput and reproducible system as an alternative to traditional linear chamber systems for exercising zebrafish and similarly sized fishes.
Publisher: Proceedings of the National Academy of Sciences
Date: 20-08-2012
Abstract: Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1 , a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311 . Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.
Publisher: Frontiers Media SA
Date: 04-03-2021
DOI: 10.3389/FCELL.2021.643603
Abstract: DNA methylation predominantly occurs at CG dinucleotides in vertebrate genomes however, non-CG methylation (mCH) is also detectable in vertebrate tissues, most notably in the nervous system. In mammals it is well established that mCH is targeted to CAC trinucleotides by DNMT3A during nervous system development where it is enriched in gene bodies and associated with transcriptional repression. Nevertheless, the conservation of developmental mCH accumulation and its deposition by DNMT3A is largely unexplored and has yet to be functionally demonstrated in other vertebrates. In this study, by analyzing DNA methylomes and transcriptomes of zebrafish brains, we identified enrichment of mCH at CAC trinucleotides (mCAC) at defined transposon motifs as well as in developmentally downregulated genes associated with developmental and neural functions. We further generated and analyzed DNA methylomes and transcriptomes of developing zebrafish larvae and demonstrated that, like in mammals, mCH accumulates during post-embryonic brain development. Finally, by employing CRISPR/Cas9 technology, we unraveled a conserved role for Dnmt3a enzymes in developmental mCAC deposition. Overall, this work demonstrates the evolutionary conservation of developmental mCH dynamics and highlights the potential of zebrafish as a model to study mCH regulation and function during normal and perturbed development.
Publisher: Oxford University Press
Date: 08-01-2020
DOI: 10.1093/OXFORDHB/9780190860509.013.8
Abstract: Chronic pain is a significant public health problem, affecting 20–25% of the global population, and there is a clear need for more specific and effective therapeutics. To achieve this, a comprehensive understanding of the underlying mechanisms and molecular machinery driving pain-related diseases is required. The definition of pain as an “unpleasant sensory and emotional experience” associated with tissue injury is innately anthropomorphic, the emotional element being difficult to reconcile in nonhuman organisms. Even simple invertebrates are nevertheless capable of nociception, the neural processing of noxious stimuli. With the significant advantages of simpler nervous systems, experimental tractability, and a high level of conservation, they have a major role to play in advancing our understanding. This chapter reviews our current molecular- and circuit-level understanding of nociception in two of the most widely used invertebrate experimental models, the nematode Caenorhabditis elegans and the fly Drosophila melanogaster. In particular, it summarizes the molecules, cells, and circuits that contribute to nociception in response to erse noxious stimuli in these model organisms and the behavioral paradigms that we can harness to study them. The chapter discusses how mechanistic insights gained from these experimental systems can improve our understanding of pain in humans.
Publisher: Rockefeller University Press
Date: 16-10-2006
Abstract: Fibulin is a broadly conserved component of the extracellular matrix (ECM). Previous studies have shown that Caenorhabditis elegans FIBULIN-1 (FBL-1) controls the width of the gonad (Hesselson, D., C. Newman, K.W. Kim, and J. Kimble. 2004. Curr. Biol. 14:2005–2010 Kubota, Y., R. Kuroki, and K. Nishiwaki. 2004. Curr. Biol. 14:2011–2018 Muriel, J.M., C. Dong, H. Hutter, and B.E. Vogel. 2005. Development. 132: 4223–4234). In this study, we report that FBL-1 also controls developmental growth and that one isoform of fibulin-1, called FBL-1C, controls both functions by distinct mechanisms. A large FBL-1C fragment, including both epidermal growth factor (EGF) and fibulin-type C domains, is responsible for constraining gonadal width, but a much smaller fragment containing only two complete EGF repeats (EGF1-2C+) is critical for developmental growth. We suggest that the larger fragment serves a scaffolding function to stabilize the basement membrane and that the smaller fragment provides a regulatory function at the cell surface or within the ECM to control growth.
Publisher: Cold Spring Harbor Laboratory
Date: 14-02-2023
DOI: 10.1101/2023.02.13.528235
Abstract: COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood 1,2 . To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium 3 , and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions. In vivo , authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.
Publisher: MDPI AG
Date: 19-10-2019
DOI: 10.3390/IJMS20205189
Abstract: Regenerative capacity varies greatly between species. Mammals are limited in their ability to regenerate damaged cells, tissues and organs compared to organisms with robust regenerative responses, such as zebrafish. The regeneration of zebrafish tissues including the heart, spinal cord and retina requires foxp3a+ zebrafish regulatory T cells (zTregs). However, it remains unclear whether the muted regenerative responses in mammals are due to impaired recruitment and/or function of homologous mammalian regulatory T cell (Treg) populations. Here, we explore the possibility of enhancing zTreg recruitment with pharmacological interventions using the well-characterized zebrafish tail utation model to establish a high-throughput screening platform. Injury-infiltrating zTregs were transgenically labelled to enable rapid quantification in live animals. We screened the NIH Clinical Collection (727 small molecules) for modulators of zTreg recruitment to the regenerating tissue at three days post-injury. We discovered that the dopamine agonist pramipexole, a drug currently approved for treating Parkinson’s Disease, specifically enhanced zTreg recruitment after injury. The dopamine antagonist SCH-23390 blocked pramipexole activity, suggesting that peripheral dopaminergic signaling may regulate zTreg recruitment. Similar pharmacological approaches for enhancing mammalian Treg recruitment may be an important step in developing novel strategies for tissue regeneration in humans.
Publisher: Wiley
Date: 11-2022
DOI: 10.1002/ECE3.9511
Abstract: The obesity epidemic, largely driven by the accessibility of ultra‐processed high‐energy foods, is one of the most pressing public health challenges of the 21st century. Consequently, there is increasing concern about the impacts of diet‐induced obesity on behavior and cognition. While research on this matter continues, to date, no study has explicitly investigated the effect of obesogenic diet on variance and covariance (correlation) in behavioral traits. Here, we examined how an obesogenic versus control diet impacts means and (co‐)variances of traits associated with body condition, behavior, and cognition in a laboratory population of ~160 adult zebrafish ( Danio rerio ). Overall, an obesogenic diet increased variation in several zebrafish traits. Zebrafish on an obesogenic diet were significantly heavier and displayed higher body weight variability fasting blood glucose levels were similar between control and treatment zebrafish. During behavioral assays, zebrafish on the obesogenic diet displayed more exploratory behavior and were less reactive to video stimuli with conspecifics during a personality test, but these significant differences were sex‐specific. Zebrafish on an obesogenic diet also displayed repeatable responses in aversive learning tests whereas control zebrafish did not, suggesting an obesogenic diet resulted in more consistent, yet impaired, behavioral responses. Where behavioral syndromes existed (inter‐class correlations between personality traits), they did not differ between obesogenic and control zebrafish groups. By integrating a multifaceted, holistic approach that incorporates components of (co‐)variances, future studies will greatly benefit by quantifying neglected dimensions of obesogenic diets on behavioral changes.
Publisher: The Company of Biologists
Date: 06-2021
DOI: 10.1242/JEB.240846
Abstract: Aversive learning – avoiding certain situations based on negative experiences – can profoundly increase fitness in animal species, yet no studies have systematically quantified its repeatability. Therefore, we assessed the repeatability of aversive learning by conditioning approximately 100 zebrafish (Danio rerio) to avoid a colour cue associated with a mild electric shock. Across eight different colour conditions, zebrafish did not show consistent in idual differences in aversive learning (R=0.04). Within conditions, when zebrafish were conditioned to the same colour, blue conditioning was more repeatable than green conditioning (R=0.15 and R=0.02). Overall, aversive learning responses of zebrafish were weak and variable. We speculate that the effect of aversive learning might have been too weak to quantify consistent in idual differences, or directional selection might have eroded additive genetic variance. We also discuss how confounded repeatability assays and publication bias could have inflated estimates of repeatability in the literature.
Publisher: Elsevier BV
Date: 02-2017
Publisher: The Company of Biologists
Date: 15-04-2015
DOI: 10.1242/DEV.117911
Abstract: The interconversion of cell lineages via transdifferentiation is an adaptive mode of tissue regeneration and an appealing therapeutic target. However, its clinical exploitation is contingent upon the discovery of contextual regulators of cell fate acquisition and maintenance. In murine models of diabetes, glucagon-secreting alpha cells transdifferentiate into insulin-secreting beta cells following targeted beta cell depletion, regenerating the form and function of the pancreatic islet. However, the molecular triggers of this mode of regeneration are unknown. Here, using lineage-tracing assays in a transgenic zebrafish model of beta cell ablation, we demonstrate conserved plasticity of alpha cells during islet regeneration. In addition, we show that glucagon expression is upregulated after injury. Through gene knockdown and rescue approaches, we also find that peptides derived from the glucagon gene are necessary for alpha-to-beta cell fate switching. Importantly, whereas beta cell neogenesis was stimulated by glucose, alpha-to-beta cell conversion was not, suggesting that transdifferentiation is not mediated by glucagon/GLP-1 control of hepatic glucose production. Overall, this study supports the hypothesis that alpha cells are an endogenous reservoir of potential new beta cells. It further reveals that glucagon plays an important role in maintaining endocrine cell homeostasis through feedback mechanisms that govern cell fate stability.
Publisher: Research Square Platform LLC
Date: 30-01-2023
DOI: 10.21203/RS.3.RS-2473300/V1
Abstract: Background Respiratory diseases are the 2 nd leading cause of death globally. The current treatments for chronic lung diseases are only supportive. Very few new classes of therapeutics have been introduced for lung diseases in the last 40 years, due to the lack of reliable lung models that enable rapid, cost-effective, and high-throughput testing. To accelerate the development of new therapeutics for lung diseases, we established two classes of lung-mimicking models: (i) healthy, and (ii) diseased lungs – COPD. Methods To establish models that mimic the lung complexity to different extents, we used five design components: (i) cell type, (ii) membrane structure/constitution, (iii) environmental conditions, (iv) cellular arrangement, (v) substrate, matrix structure and composition. To determine whether the lung models are reproducible and reliable, we developed a quality control (QC) strategy, which integrated the real-time and end-point quantitative and qualitative measurements of cellular barrier function, permeability, tight junctions, tissue structure, tissue composition, and cytokine secretion. Results The healthy model is characterised by (i) continuous tight junctions, (ii) physiological cellular barrier function, (iii) a full thickness epithelium composed of multiple cell layers, and (iv) the presence of ciliated cells and goblet cells. Meanwhile, the disease model emulates human COPD disease: (i) dysfunctional cellular barrier function, (ii) depletion of ciliated cells, and (ii) overproduction of goblet cells. The models developed here have multiple competitive advantages when compared with existing in vitro lung models: (i) the macroscale enables multimodal and correlative characterisation of the same model system, (ii) the use of cells derived from patients that enables the creation of in idual models for each patient for personalised medicine, (iii) the use of an extracellular matrix proteins interface, which promotes physiological cell adhesion and differentiation, (iv) media microcirculation that mimics the dynamic conditions in human lungs. Conclusion Our model can be utilised to test safety, efficacy, and superiority of new therapeutics as well as to test toxicity and injury induced by inhaled pollution or pathogens. It is envisaged that these models can also be used to test the protective function of new therapeutics for high-risk patients or workers exposed to occupational hazards.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 09-04-2021
Abstract: Although humans show minimal regenerative capability, zebrafish can regenerate their hearts through a mechanism whereby heart muscle cells (cardiomyocytes) revert to a less mature state and then proliferate to replace the damaged tissue. Ogawa et al. show that Krüppel-like factor 1 (Klf1/Eklf), a transcription factor well known for its role in red blood cell development, is an essential factor for heart regeneration in zebrafish. Klf1 is specifically expressed in cardiomyocytes after injury, and its activation is sufficient to stimulate new cardiomyocyte production without injury. This potent effect is achieved through reprogramming of gene networks regulating cardiomyocyte differentiation and mitochondrial metabolism. Science , this issue p. 201
Publisher: Rockefeller University Press
Date: 29-06-2015
DOI: 10.1084/JEM.20150218
Abstract: The nuclear factor κB (NF-κB) pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic β cell dysfunction in the metabolic syndrome. Whereas canonical NF-κB signaling is well studied, there is little information on the ergent noncanonical NF-κB pathway in the context of pancreatic islet dysfunction. Here, we demonstrate that pharmacological activation of the noncanonical NF-κB–inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. We identify NIK as a critical negative regulator of β cell function, as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of noncanonical NF-κB components p100 to p52, and accumulation of RelB. TNF and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive β cell–intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the noncanonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to β cell failure. These studies reveal that NIK contributes a central mechanism for β cell failure in diet-induced obesity.
Publisher: Mary Ann Liebert Inc
Date: 04-2018
Abstract: An emergent field of animal personality necessitates a method for repeated high-throughput quantification of behavioral traits across contexts. In this study, we have developed an automated video stimulus approach to sequentially present different contexts relevant to five "personality" traits (exploration, boldness, neophobia, aggression, and sociability), successfully quantifying repeatable trait measurements in multiple in iduals simultaneously. Although our method is designed to quantify personality traits in zebrafish, our approach can accommodate the quantification of other behaviors, and could be customized for other species. All digital materials and detailed protocols are publicly available online for researchers to freely use and modify.
Publisher: Elsevier BV
Date: 04-2017
DOI: 10.1016/J.CHEMBIOL.2017.03.005
Abstract: Stimulating autophagy is a promising therapeutic strategy for slowing the progression of neurodegenerative disease. Neurons are insensitive to current approaches based on mTOR inhibition for activating autophagy, and instead may rely on the Parkinson's disease-associated proteins PINK1 and PARKIN to activate the autophagy-lysosomal pathway in response to mitochondrial damage. We developed a multifactorial zebrafish drug-screening platform combining Pink1 deficiency with an environmental toxin to compromise mitochondrial function and trigger dopaminergic neuron loss. Using a phenotypic screening strategy, we identified a series of piperazine phenothiazines, including trifluoperazine, which rescued Pink1 deficiency by activating autophagy selectively in stressed zebrafish and human cells. We show that trifluoperazine acts downstream of, or parallel to, PINK1/PARKIN to stimulate transcription factor EB nuclear translocation and the expression of autophagy-lysosomal target genes. These data suggest that stress-dependent pharmacological reactivation of autophagy could prevent the loss of vulnerable neurons to slow neurodegeneration.
Publisher: Elsevier BV
Date: 07-2013
Publisher: Springer Science and Business Media LLC
Date: 30-04-2019
DOI: 10.1038/S41467-019-09681-1
Abstract: The box jellyfish Chironex fleckeri is extremely venomous, and envenoming causes tissue necrosis, extreme pain and death within minutes after severe exposure. Despite rapid and potent venom action, basic mechanistic insight is lacking. Here we perform molecular dissection of a jellyfish venom-induced cell death pathway by screening for host components required for venom exposure-induced cell death using genome-scale lenti-CRISPR mutagenesis. We identify the peripheral membrane protein ATP2B1, a calcium transporting ATPase, as one host factor required for venom cytotoxicity. Targeting ATP2B1 prevents venom action and confers long lasting protection. Informatics analysis of host genes required for venom cytotoxicity reveal pathways not previously implicated in cell death. We also discover a venom antidote that functions up to 15 minutes after exposure and suppresses tissue necrosis and pain in mice. These results highlight the power of whole genome CRISPR screening to investigate venom mechanisms of action and to rapidly identify new medicines.
Publisher: Springer Science and Business Media LLC
Date: 10-11-2017
DOI: 10.1038/S41467-017-01325-6
Abstract: Forces play erse roles in vascular development, homeostasis and disease. VE-cadherin at endothelial cell-cell junctions links the contractile acto-myosin cytoskeletons of adjacent cells, serving as a tension-transducer. To explore tensile changes across VE-cadherin in live zebrafish, we tailored an optical biosensor approach, originally established in vitro. We validate localization and function of a VE-cadherin tension sensor (TS) in vivo. Changes in tension across VE-cadherin observed using ratio-metric or lifetime FRET measurements reflect acto-myosin contractility within endothelial cells. Furthermore, we apply the TS to reveal biologically relevant changes in VE-cadherin tension that occur as the dorsal aorta matures and upon genetic and chemical perturbations during embryonic development.
Publisher: American Chemical Society (ACS)
Date: 09-07-2019
DOI: 10.1021/ACS.ORGLETT.9B01838
Abstract: The first approaches to the 10'-anthronyl-2-anthraquinone skeleton have been devised, allowing two syntheses of the marine natural product albopunctatone. Both routes involve regioselective addition of a nucleophilic masked anthraquinone to a protected chrysazin derivative the best affords albopunctatone in five steps and 35% overall yield. Albopunctatone exhibits potent inhibitory activity against
Publisher: American Chemical Society (ACS)
Date: 11-10-2022
DOI: 10.1021/ACSSYNBIO.2C00460
Abstract: Directed evolution uses cycles of gene ersification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or "VEGAS") has been described. Here we report that the VEGAS system has major limitations that preclude its use for directed evolution. The deconstructed Sindbis virus (SINV) genome that comprises the VEGAS system could no longer promote Sindbis structural gene (SSG)-dependent viral replication. Moreover, viral particles generated using the VEGAS system rapidly lost the target directed evolution transgene, and instead, "cheater" particles, primarily containing RNA encoding SINV structural components, arose. By sequencing, we found that this contamination came from RNA provided during initial SINV packaging, not RNA derived from the VEGAS system. Of note, both the structural RNA and target transgenes used in the VEGAS system contain viral packaging sequences. The impact of SINV "cheater" particles could be potentially overcome in the context of a robust VEGAS circuit, but since SSG complementation is also defective in the VEGAS system, selection for authentic evolution products is not currently possible. Similar results have been obtained in independent laboratories. Taken together, these results show that the VEGAS system does not work as described and, without significant redesign, cannot be used for mammalian directed evolution c aigns.
Publisher: Elsevier BV
Date: 04-2011
Publisher: Elsevier BV
Date: 2022
Publisher: Oxford University Press (OUP)
Date: 2016
DOI: 10.1093/EEP/DVV014
Location: United States of America
Start Date: 2003
End Date: 2006
Funder: Canadian Institutes of Health Research
View Funded ActivityStart Date: 03-2022
End Date: 03-2025
Amount: $528,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 2020
End Date: 06-2023
Amount: $473,782.00
Funder: Australian Research Council
View Funded Activity