ORCID Profile
0000-0002-7434-926X
Current Organisation
Australian National University
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Central Nervous System | Neurosciences
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in Psychology and Cognitive Sciences |
Publisher: Springer Science and Business Media LLC
Date: 26-10-2015
Publisher: Springer Science and Business Media LLC
Date: 29-07-2015
Publisher: Cold Spring Harbor Laboratory
Date: 16-03-2022
DOI: 10.1101/2022.03.15.484427
Abstract: Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the BH using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans- DNA cleavage activity. We show that helix 1 and not the BH is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the BH upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.
Publisher: Springer Science and Business Media LLC
Date: 27-04-2020
Publisher: Springer Science and Business Media LLC
Date: 26-02-2019
DOI: 10.1038/S41598-019-39142-0
Abstract: Editing in idual nucleotides is a crucial component for validating genomic disease association. It is currently h ered by CRISPR-Cas-mediated “base editing” being limited to certain nucleotide changes, and only achievable within a small window around CRISPR-Cas target sites. The more versatile alternative, HDR (homology directed repair), has a 3-fold lower efficiency with known optimization factors being largely immutable in experiments. Here, we investigated the variable efficiency-governing factors on a novel mouse dataset using machine learning. We found the sequence composition of the single-stranded oligodeoxynucleotide (ssODN), i.e. the repair template, to be a governing factor. Furthermore, different regions of the ssODN have variable influence, which reflects the underlying mechanism of the repair process. Our model improves HDR efficiency by 83% compared to traditionally chosen targets. Using our findings, we developed CUNE (Computational Universal Nucleotide Editor), which enables users to identify and design the optimal targeting strategy using traditional base editing or – for-the-first-time – HDR-mediated nucleotide changes.
Publisher: Mary Ann Liebert Inc
Date: 02-2018
Publisher: Cold Spring Harbor Laboratory
Date: 08-03-2017
DOI: 10.1101/114959
Abstract: Allelic heterogeneity is a common phenomenon where a gene exhibit different phenotype depending on the nature of its genetic mutations. In the context of genes affecting malaria susceptibility, it allowed us to explore and understand the intricate host-parasite interactions during malaria infections. In this study, we described a gene encoding erythrocytic ankyrin-1 ( Ank-1 ) which exhibits allelic-dependent heterogeneous phenotypes during malaria infections. We conducted an ENU mutagenesis screen on mice and identified two Ank-1 mutations, one resulted in an amino acid substitution (MRI95845), and the other a truncated Ank-1 protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection against Plasmodium chabaudi infections. Upon further examination, the Ank-1 (MRI96570) mutation was found to inhibit intra-erythrocytic parasite maturation, whereas Ank-1 (MW95845) caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between two Ank-1 mutations. Despite the lack of direct evidence from population studies, this data further supported the protective roles of ankyrin-1 mutations in conferring malaria protection. This study also emphasized the importance of such phenomenon to achieve a better understanding of host-parasite interactions, which could be the basis of future studies.
Publisher: Cold Spring Harbor Laboratory
Date: 05-2022
DOI: 10.1101/2022.04.29.490022
Abstract: Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A. baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1β and IL-18, the recruitment of innate immune effectors against A. baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the host’s innate response and activation of the inflammasome during A. baumannii infection. In this study, we compared nine A. baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death pathway in primary macrophages and mice. We found a striking variability in survival outcomes of mice and bacterial dissemination in organs among three ATCC A. baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found a stark contrast in activation of the NLRP3 inflammasome pathway, the non-canonical caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokines IL-1β and IL-18 between A. baumannii strains. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with differential virulence to investigate the innate immune response to A. baumannii infection.
Publisher: Informa UK Limited
Date: 13-05-2013
DOI: 10.4161/CIB.23653
Publisher: Public Library of Science (PLoS)
Date: 22-04-2015
Publisher: Springer Science and Business Media LLC
Date: 30-11-2011
DOI: 10.1007/S00335-010-9302-6
Abstract: Malaria is a disease that infects over 500 million people, causing at least 1 million deaths every year, with the majority occurring in developing countries. The current antimalarial arsenal is becoming dulled due to the rapid rate of resistance of the parasite. However, in populations living in malaria-endemic regions there are many ex les of genetic-based resistance to the severe effects of the parasite Plasmodium. Defining the genetic factors behind host resistance has been an area of great scientific interest over the last few decades this review summarizes the current knowledge of the genetic loci involved. Perhaps the lessons learned from the natural variation in both the human populations and experimental mouse models of infection may pave the way for novel resistance-proof antimalarials.
Publisher: Springer Science and Business Media LLC
Date: 07-2019
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2021
DOI: 10.1101/2021.01.21.427535
Abstract: The nucleolar surveillance pathway (NSP) monitors nucleolar fidelity and responds to nucleolar stresses (i.e., inactivation of ribosome biogenesis) by mediating the inhibitory binding of ribosomal proteins (RPs) to mouse double minute 2 homolog (MDM2), a nuclear-localised E3 ubiquitin ligase, which results in p53 accumulation. Inappropriate activation of the NSP has been implicated in the pathogenesis of collection of human diseases termed “ribosomopathies”, while drugs that selectively activate the NSP are now in trials for cancer. Despite the clinical significance, the precise molecular mechanism(s) regulating the NSP remain poorly understood. Using genome-wide loss of function screens, we demonstrate the ribosome biogenesis (RiBi) axis as the most potent class of genes whose disruption stabilises p53. Furthermore, we identified a novel suite of genes critical for the NSP, including a novel mammalian protein implicated in 5S ribonucleoprotein particle (5S-RNP) biogenesis, HEATR3. By selectively disabling the NSP, we unexpectedly demonstrate that a functional NSP is required for the ability of all nuclear acting stresses tested, including DNA damage, to robustly induce p53 accumulation. Together, our data demonstrates that the NSP has evolved as the dominant central integrator of stresses that regulate nuclear p53 abundance, thus ensuring RiBi is hardwired to cellular proliferative capacity.
Publisher: Elsevier BV
Date: 03-2022
Publisher: Cold Spring Harbor Laboratory
Date: 31-08-2016
DOI: 10.1101/072587
Abstract: Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1 (MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1 (MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1 (MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection the “bystander effect”. This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1 (MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance.
Publisher: Rockefeller University Press
Date: 18-10-2018
DOI: 10.1084/JEM.20180639
Abstract: Genetic mutations account for many devastating early onset immune deficiencies. In contrast, less severe and later onset immune diseases, including in patients with no prior family history, remain poorly understood. Whole exome sequencing in two cohorts of such patients identified a novel heterozygous de novo IKBKB missense mutation (c.607G& A) in two separate kindreds in whom probands presented with immune dysregulation, combined T and B cell deficiency, inflammation, and epithelial defects. IKBKB encodes IKK2, which activates NF-κB signaling. IKK2V203I results in enhanced NF-κB signaling, as well as T and B cell functional defects. IKK2V203 is a highly conserved residue, and to prove causation, we generated an accurate mouse model by introducing the precise orthologous codon change in Ikbkb using CRISPR/Cas9. Mice and humans carrying this missense mutation exhibit remarkably similar cellular and biochemical phenotypes. Accurate mouse models engineered by CRISPR/Cas9 can help characterize novel syndromes arising from de novo germline mutations and yield insight into pathogenesis.
Publisher: Oxford University Press (OUP)
Date: 12-2018
Abstract: Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance. Acinetobacter baumannii has an exceptional ability to rapidly develop drug resistance and to adhere to abiotic surfaces, including medical equipment, significantly promoting bacterial spread and also limiting our ability to control A. baumannii infections. Consequently, A. baumannii is frequently responsible for ventilator-associated pneumonia in clinical settings. In order to develop an effective treatment strategy, understanding host-pathogen interactions during A. baumannii infection is crucial. Various A. baumannii virulence factors have been identified as targets of host innate pattern-recognition receptors, which leads to activation of downstream inflammasomes to develop inflammatory responses, and the recruitment of innate immune effectors against A. baumannii infection. To counteract host immune attack, A. baumannii regulates its expression of different virulence factors. This review summarizes the significance of mechanisms of host-bacteria interaction, as well as different bacteria and host defense mechanisms during A. baumannii infection.
Publisher: Springer Science and Business Media LLC
Date: 28-02-2018
Publisher: MDPI AG
Date: 29-09-2021
Abstract: High protein feeding has been shown to accelerate the development of type 1 diabetes in female non-obese diabetic (NOD) mice. Here, we investigated whether reducing systemic amino acid availability via knockout of the Slc6a19 gene encoding the system B(0) neutral amino acid transporter AT1 would reduce the incidence or delay the onset of type 1 diabetes in female NOD mice. Slc6a19 gene deficient NOD mice were generated using the CRISPR-Cas9 system which resulted in marked aminoaciduria. The incidence of diabetes by week 30 was 59.5% (22/37) and 69.0% (20/29) in NOD.Slc6a19+/+ and NOD.Slc6a19−/− mice, respectively (hazard ratio 0.77, 95% confidence interval 0.41–1.42 Mantel-Cox log rank test: p = 0.37). The median survival time without diabetes was 28 and 25 weeks for NOD.Slc6a19+/+ and NOD.Slc6a19−/− mice, respectively (ratio 1.1, 95% confidence interval 0.6–2.0). Histological analysis did not show differences in islet number or the degree of insulitis between wild type and Slc6a19 deficient NOD mice. We conclude that Slc6a19 deficiency does not prevent or delay the development of type 1 diabetes in female NOD mice.
Publisher: American Society for Microbiology
Date: 27-04-2021
DOI: 10.1128/MSYSTEMS.00182-21
Abstract: Malaria still causes about 400,000 deaths a year and is one of the most studied infectious diseases. The disease is studied in mice and monkeys as lab models to derive potential therapeutic intervention in human malaria.
Publisher: Public Library of Science (PLoS)
Date: 17-10-2023
Publisher: American Society of Hematology
Date: 09-2016
DOI: 10.1182/BLOOD-2015-09-666834
Abstract: AMPD3 activation reduces red blood cell half-life, which is associated with increased oxidative stress and phosphatidylserine exposure. AMPD3 activation causes malaria resistance through increased RBC turnover and increased RBC production.
Publisher: Springer Science and Business Media LLC
Date: 05-02-2020
DOI: 10.1038/S41598-020-59357-W
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Publisher: Springer Science and Business Media LLC
Date: 10-12-2018
Publisher: Cold Spring Harbor Laboratory
Date: 13-03-2019
DOI: 10.1101/576116
Abstract: Dual RNA-Seq is the simultaneous transcriptomic analysis of interacting symbionts, for ex le, in malaria. Potential cross-species interactions identified by correlated gene expression might highlight interlinked signaling, metabolic or gene regulatory pathways in addition to physically interacting proteins. Often, malaria studies address one of the interacting organisms – host or parasite – rendering the other “contamination”. Here we perform a meta-analysis using such studies for cross-species expression analysis. We screened experiments for gene expression from host and Plasmodium . Out of 171 studies in Homo sapiens, Macaca mulatta and Mus musculus , we identified 63 potential studies containing host and parasite data. While 16 studies (1950 s les) explicitly performed dual RNA-Seq, 47 (1398 s les) originally focused on one organism. We found 915 experimental replicates from 20 blood studies to be suitable for co-expression analysis and used orthologs for meta-analysis across different host-parasite systems. Centrality metrics from the derived gene expression networks correlated with gene essentiality in the parasites. We found indications of host immune response to elements of the Plasmodium protein degradation system, an antimalarial drug target. We identified well-studied immune responses in the host with our co-expression networks as our approach recovers known broad processes interlinked between hosts and parasites in addition to in idual host and parasite protein associations. The set of core interactions represents commonalities between human malaria and its model systems for prioritization in laboratory experiments. Our approach might also allow insights into the transferability of model systems for different pathways in malaria studies. Malaria still causes about 400,000 deaths a year and is one the most studied infectious diseases. The disease is studied in mice and monkeys as lab models to derive potential therapeutic intervention in human malaria. Interactions between Plasmodium spp. and its hosts are either conserved across different host-parasite systems or idiosyncratic to those systems. Here we use correlation of gene expression from different RNA-Seq studies to infer common host-parasite interactions across human, mouse and monkey studies. We, firstly, find a set of very conserved interactors, worth further scrutiny in focussed laboratory experiments. Secondly, this work might help assess to which extent experiments and knowledge on different pathways can be transferred from models to humans for potential therapy.
Publisher: American Chemical Society (ACS)
Date: 04-2021
Publisher: Public Library of Science (PLoS)
Date: 03-04-2014
Publisher: Springer Science and Business Media LLC
Date: 17-05-2019
DOI: 10.1038/S41467-019-10242-9
Abstract: Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.
Publisher: Springer Science and Business Media LLC
Date: 29-07-2022
DOI: 10.1038/S41467-022-32127-0
Abstract: Inflammasomes are cytosolic signaling complexes capable of sensing microbial ligands to trigger inflammation and cell death responses. Here, we show that guanylate-binding proteins (GBPs) mediate pathogen-selective inflammasome activation. We show that mouse GBP1 and GBP3 are specifically required for inflammasome activation during infection with the cytosolic bacterium Francisella novicida . We show that the selectivity of mouse GBP1 and GBP3 derives from a region within the N-terminal domain containing charged and hydrophobic amino acids, which binds to and facilitates direct killing of F. novicida and Neisseria meningitidis , but not other bacteria or mammalian cells. This pathogen-selective recognition by this region of mouse GBP1 and GBP3 leads to pathogen membrane rupture and release of intracellular content for inflammasome sensing. Our results imply that GBPs discriminate between pathogens, confer activation of innate immunity, and provide a host-inspired roadmap for the design of synthetic antimicrobial peptides that may be of use against emerging and re-emerging pathogens.
Publisher: Public Library of Science (PLoS)
Date: 25-05-2012
Publisher: Cold Spring Harbor Laboratory
Date: 06-12-2016
DOI: 10.1101/091678
Abstract: A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci ( Sptb , Tet-1 , Tet-2 and Tet-3 ) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’- phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected s les. Together our findings indicate that we failed to edit using NgAgo.
Publisher: Cold Spring Harbor Laboratory
Date: 25-10-2022
DOI: 10.1101/2022.10.24.513490
Abstract: Multidrug resistant (MDR) Acinetobacter baumannii are of major concern worldwide due to their resistance to last resort carbapenem and polymyxin antibiotics. To develop an effective treatment strategy, it is critical to better understand how an A. baumannii MDR bacterium interacts with its mammalian host. Pattern-recognition receptors sense microbes, and activate the inflammasome pathway, leading to pro-inflammatory cytokine production and programmed cell death. Here, we found that MDR A. baumannii activate the NLRP3 inflammasome complex predominantly via the non-canonical caspase-11-dependent pathway. We show that caspase-11-deficient mice are protected from a virulent MDR A. baumannii strain by maintaining a balance between protective and deleterious inflammation via IL-1. Caspase-11-deficient mice also compromise between effector cell recruitment, phagocytosis, and programmed cell death in the lung during infection. Importantly, we found that cytosolic immunity - mediated by guanylate-binding protein 1 (GBP1) and type I interferon signalling - orchestrates caspase-11-dependent inflammasome activation. This exerts a bactericidal activity against carbapenem- and colistin-resistant, lipooligosaccharide (LOS)- deficient bacteria. Together, our results suggest that developing therapeutic strategies targeting GBP1 might pave the way as a host-directed therapy to overcome multidrug resistance in A. baumannii infection.
Publisher: UPV/EHU Press
Date: 2009
Abstract: Recurrent Spontaneous Abortion (RSA) is a frequent pathology affecting 1 to 5% of couples. In approximately 50 % of cases, the aetiology is unknown suggesting a subtle interaction between genetic and environmental factors. Previous attempts to describe genetic factors using the candidate gene approach have been relatively unsuccessful due to the physiological, cellular and genetic complexity of mammalian reproduction. Indeed, fertility can be considered as a quantitative feature resulting from the interaction of genetic, epigenetic and environmental factors. Herein, we identified Quantitative Trait Loci (QTL) associated with erse embryonic lethality phenotypes and the subsequent embryonic resorption in 39 inter-specific recombinant congenic mice strains, using in vivo ultrasound bio-microscopy. The short chromosomal intervals related to the phenotypes will facilitate the study of a restricted number of candidate genes which are potentially dysregulated in patients affected by RSA.
Publisher: Cold Spring Harbor Laboratory
Date: 05-10-2020
DOI: 10.1101/2020.10.02.324079
Abstract: The Plasmodium parasites that cause malaria are adept at developing resistance to antimalarial drugs, necessitating the search for new antiplasmodials. Although several amide analogs of pantothenate (pantothenamides) show potent antiplasmodial activity, hydrolysis by pantetheinases (or vanins) present in blood rapidly inactivates them. We report herein the facile synthesis and biological activity of a small library of pantothenamide analogs in which the labile amide group is replaced with a variety of heteroaromatic rings. Several of the new analogs display antiplasmodial activity in the nanomolar range against P. falciparum and/or P. knowlesi in the presence of pantetheinase. A previously reported triazole and an isoxazole derivative presented here were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro . Although we show here that the two compounds fail to suppress proliferation of P. berghei in vivo , pharmacokinetic and contact time data presented provide a benchmark for the compound profile required to achieve antiplasmodial activity in mice and should facilitate lead optimization.
Publisher: Wiley
Date: 20-08-2015
DOI: 10.1111/VCP.12276
Abstract: In chronic kidney disease (CKD), anemia and hypertension are significant co-morbidities that contribute to cardiovascular and renal disease progression. The purpose of the study was to identify correlations between changes in hematologic variables against markers of renal function, blood pressure, and erythropoietin (EPO) in a naturally occurring hypertensive model of CKD, the Lewis polycystic kidney (LPK) rat. Complete blood count, systolic blood pressure, urea and creatinine concentration, urinary protein to creatinine ratio, and plasma EPO concentration were determined in control Lewis (n = 51) and LPK rats (n = 56) aged 6-24 weeks. Renal EPO gene expression and RBC osmotic fragility were also documented. Hematopoiesis in spleen and bone marrow were assessed. Lewis polycystic kidney rats had increasing urea and creatinine concentrations, concurrent with the development of a nonregenerative normocytic/normochromic anemia and hypertension, with a significant negative correlation between both HGB and HCT with urea concentration and blood pressure (P < .01). HCT was also significantly negatively correlated with creatinine concentration (P = .014). WBC was significantly negatively correlated with urea (P < .01). Plasma EPO concentration was increased and renal EPO mRNA expression was significantly upregulated in LPK animals. The former was significantly positively correlated with blood pressure and platelet count (P < .05). RBC osmotic fragility was normal in LPK rats and there was no evidence for increased RBC elimination or extramedullary hematopoiesis. Marked anemia in the LPK CKD rodent model in the presence of elevated EPO suggests inefficient erythropoiesis that is correlated with plasma urea concentration and blood pressure.
Publisher: Public Library of Science (PLoS)
Date: 11-2022
DOI: 10.1371/JOURNAL.PONE.0277019
Abstract: Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance globally. A . baumannii has an exceptional ability to rapidly develop drug resistance. It is frequently responsible for ventilator-associated pneumonia in clinical settings and inflammation resulting in severe sepsis. The inflammatory response is mediated by host pattern-recognition receptors and the inflammasomes. Inflammasome activation triggers inflammatory responses, including the secretion of the pro-inflammatory cytokines IL-1β and IL-18, the recruitment of innate immune effectors against A . baumannii infection, and the induction programmed cell death by pyroptosis. An important knowledge gap is how variation among clinical isolates affects the host’s innate response and activation of the inflammasome during A . baumannii infection. In this study, we compared nine A . baumannii strains, including clinical locally-acquired isolates, in their ability to induce activation of the inflammasome and programmed cell death in primary macrophages, epithelial lung cell line and mice. We found a variation in survival outcomes of mice and bacterial dissemination in organs among three commercially available A . baumannii strains, likely due to the differences in virulence between strains. Interestingly, we found variability among A . baumannii strains in activation of the NLRP3 inflammasome, non-canonical Caspase-11 pathway, plasmatic secretion of the pro-inflammatory cytokine IL-1β and programmed cell death. Our study highlights the importance of utilising multiple bacterial strains and clinical isolates with different virulence to investigate the innate immune response to A . baumannii infection.
Publisher: Wiley
Date: 09-07-2020
Publisher: American Society for Microbiology
Date: 11-2015
DOI: 10.1128/IAI.00926-15
Abstract: The treatment of iron deficiency in areas of high malaria transmission is complicated by evidence which suggests that iron deficiency anemia protects against malaria, while iron supplementation increases malaria risk. Iron deficiency anemia results in an array of pathologies, including reduced systemic iron bioavailability and abnormal erythrocyte physiology however, the mechanisms by which these pathologies influence malaria infection are not well defined. In the present study, the response to malaria infection was examined in a mutant mouse line, Tfrc MRI24910 , identified during an N -ethyl- N -nitrosourea (ENU) screen. This line carries a missense mutation in the gene for transferrin receptor 1 (TFR1). Heterozygous mice exhibited reduced erythrocyte volume and density, a phenotype consistent with dietary iron deficiency anemia. However, unlike the case in dietary deficiency, the erythrocyte half-life, mean corpuscular hemoglobin concentration, and intraerythrocytic ferritin content were unchanged. Systemic iron bioavailability was also unchanged, indicating that this mutation results in erythrocytic iron deficiency without significantly altering overall iron homeostasis. When infected with the rodent malaria parasite Plasmodium chabaudi adami , mice displayed increased parasitemia and succumbed to infection more quickly than their wild-type littermates. Transfusion of fluorescently labeled erythrocytes into malaria parasite-infected mice demonstrated an erythrocyte-autonomous enhanced survival of parasites within mutant erythrocytes. Together, these results indicate that TFR1 deficiency alters erythrocyte physiology in a way that is similar to dietary iron deficiency anemia, albeit to a lesser degree, and that this promotes intraerythrocytic parasite survival and an increased susceptibility to malaria in mice. These findings may have implications for the management of iron deficiency in the context of malaria.
Publisher: Cold Spring Harbor Laboratory
Date: 03-2023
DOI: 10.1101/2023.02.28.530512
Abstract: Genome editing through the development of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) – Cas technology has revolutionized many fields in biology. Beyond Cas9 nucleases, Cas12a (formerly Cpf1) has emerged as a promising alternative to Cas9 for editing AT-rich genomes. Despite the promises, guide RNA efficiency prediction through computational tools search still lacks accuracy. Through a computational meta-analysis, here we report that Cas12a target and off-target cleavage behaviour are factor of nucleotide bias combined with nucleotide mismatches relative to the protospacer adjacent motif (PAM) site. These features helped to train a machine learning random forest algorithm to improve the accuracy by at least 15% to existing algorithms to predict guide RNA efficiency for Cas12a enzyme. Despite the progresses, our report underscores the need for more representative datasets and further benchmarking to reliably and accurately predict guide RNA efficiency and off-target effects for Cas12a enzymes.
Publisher: Cold Spring Harbor Laboratory
Date: 27-03-2019
DOI: 10.1101/589242
Abstract: An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel nonsense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as Pbgd MRI58155 . Heterozygote Pbgd MRI58155 mice exhibited approximately 50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was approximately 10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi , which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd -deficient mice infected with P. berghei , which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20-50%) reduced levels of PBGD. Furthermore, the growth of Pbgd -null P. falciparum and Pbgd -null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd -deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum , but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection. The causative agent of malaria, Plasmodium , adopts a parasitic lifestyle during erythrocyte infection, and as such relies on host cell factors for its survival and growth. Host-encoded mutations that alter the availability of these factors confer disease resistance, including several well-known genetic erythrocyte abnormalities that have arisen due to the historical evolutionary pressure of malaria. This study identified in mice a novel malaria resistance-conferring host mutation in the heme biosynthesis enzyme, porphobilinogen deaminase (PBGD), and compared the relative requirements by Plasmodium for the host versus parasite-encoded forms of PBGD in both in vivo and in vitro settings. The findings demonstrated that parasite PBGD was dispensable, but that the host enzyme was important specifically during in vivo infection by P. chabaudi , and collectively suggest that Plasmodium requires a certain threshold of the enzyme to sustain its intraerythrocytic growth. Plasmodium may therefore be vulnerable to other interventions that limit host PBGD activity.
Publisher: Springer Science and Business Media LLC
Date: 06-02-2020
DOI: 10.1038/S41467-020-14534-3
Abstract: Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome.
Publisher: MyJove Corporation
Date: 05-04-2015
DOI: 10.3791/52736
Publisher: Cold Spring Harbor Laboratory
Date: 07-11-2018
DOI: 10.1101/464610
Abstract: Editing in idual nucleotides is a crucial component for validating genomic disease association. It currently is h ered by CRISPR-Cas-mediated “base editing” being limited to certain nucleotide changes, and only achievable within a small window around CRISPR-Cas target sites. The more versatile alternative, HDR (homology directed repair), has a 4-fold lower efficiency with known optimization factors being largely immutable in experiments. Here, we investigated the variable efficiency-governing factors on a novel mouse dataset using machine learning. We found the sequence composition of the repair template (ssODN) to be a governing factor, where different regions of the ssODN have variable influence, which reflects the underlying biophysical mechanism. Our model improves HDR efficiency by 83% compared to traditionally chosen targets. Using our findings, we develop CUNE (Computational Universal Nucleotide Editor), which enables users to identify and design the optimal targeting strategy using traditional base editing or – for-the-first-time – HDR-mediated nucleotide changes. CUNE can be run via the web at: une
Publisher: Research Square Platform LLC
Date: 27-01-2021
DOI: 10.21203/RS.3.RS-152145/V1
Abstract: While circumstantial evidence supports enhanced TLR7 signaling as a mechanism of human systemic autoimmune disease, we have lacked the proof afforded by lupus-causing TLR7 gene variants. Here we describe monogenic human systemic lupus erythematosus (SLE) caused by TLR7 gain-of-function. We identified a de novo , novel, missense TLR7 Y264H variant in a child with severe lupus and additional novel or rare variants in probands with interferonopathies or systemic autoimmunity (Aicardi Goutieres Sd, SLE, Sjogren’s Sd, and juvenile idiopathic arthritis). The variants increased NF-κB and IFN-β activity and the de novo TLR7 Y264H variant was sufficient to cause lupus when introduced in mice. We show that constitutive TLR7 signaling drives aberrant survival of BCR-activated B cells that would otherwise die, and accumulation of CD11c + age-associated B cells and germinal center (GC) B cells in a B cell-intrinsic manner. Follicular and extrafollicular helper T-cells were also increased but these phenotypes were cell-extrinsic. MyD88-deficiency rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous GC formation in mice carrying the TLR7 Y264H variant, we show that TLR7-driven lupus was not ameliorated when the TLR7 Y264H mice were made GC-deficient suggesting extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 for human SLE pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.
Publisher: Cold Spring Harbor Laboratory
Date: 29-04-2023
DOI: 10.1101/2023.04.27.538529
Abstract: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3 - a negative regulator of cytokine and growth factor receptor signaling – harbors rare coding variants in over 5% of SLE patients. Here we show that unlike the variant found exclusively in healthy controls, most SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles. Generation of two mouse lines carrying variants orthologous to those found in patients revealed SH2B3 is important to limit the numbers of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk. Zhang et al . reveal a role for hypomorphic SH2B3 in lupus risk. The study shows rare and damaging variants identified in lupus patients enable breach of B cell immune tolerance checkpoints and suggests involvement for dysregulated IL-4R signaling and BAFF-R expression.
Publisher: Cold Spring Harbor Laboratory
Date: 16-03-2022
DOI: 10.1101/2022.03.14.484124
Abstract: The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in in idual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in in idual RNA molecules from the same s le, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same s le reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
Publisher: Emerald
Date: 16-11-2015
Abstract: – Networks and networking are ubiquitous concepts in tourism, their importance appreciated by scholars and practitioners. Tourism research has developed elaborate methods and concepts to grasp the numerous variants of tourism networks and to gain insights into their governance. In particular, Association Internationale D’Experts Scientifiques Du Tourisme (AIEST) and its official journal, Tourism Review , have made significant contributions to the achievements in this research area. After approximately two decades of intensive research on tourism networks, it is appropriate to pause for a moment to critically assess the results achieved, to compare them with partly old, partly newly, emerging real-world challenges, and to explore future directions. – This paper provides a selective and critical overview of the state-of-the-art in research on governing networks in tourism. This overview of eight major achievements is combined with an exploratory, comparative analysis of qualitative interviews with tourism practitioners. – Considering the two sources mentioned above, the study derives seven suggestions for future directions in research on network governance in tourism. These relate to the big picture of tourism governance, e-governance, disparities within networks, negative aspects of networking, dynamism of networks, network moderators and means of network steering. – Due to its design, the paper is uniquely able to compare real-world issues with up-to-date theoretical achievements, and will contribute to bringing them closer together in future approaches. Hence, it is relevant for both academic readers and practitioners.
Publisher: Springer Science and Business Media LLC
Date: 23-04-2019
DOI: 10.1038/S41598-019-42782-X
Abstract: Plasmodium falciparum malaria causes half a million deaths per year, with up to 9% of this mortality caused by cerebral malaria (CM). One of the major processes contributing to the development of CM is an excess of host inflammatory cytokines. Recently K+ signaling has emerged as an important mediator of the inflammatory response to infection we therefore investigated whether mice carrying an ENU induced activation of the electroneutral K+ channel KCC1 had an altered response to Plasmodium berghei . Here we show that Kcc1 M935K/M935K mice are protected from the development of experimental cerebral malaria, and that this protection is associated with an increased CD4+ and TNFa response. This is the first description of a K+ channel affecting the development of experimental cerebral malaria.
Publisher: Public Library of Science (PLoS)
Date: 14-03-2019
Publisher: Wiley
Date: 20-02-2019
Abstract: Mutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE-affected mice further supported the Th17 cell-specific migration defect, however, DOCK8-deficient Th17 cells expressed normal Th17 cell-specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias.
Publisher: Oxford University Press (OUP)
Date: 09-0001
Abstract: Allelic heterogeneity is a common phenomenon where a gene exhibits a different phenotype depending on the nature of its genetic mutations. In the context of genes affecting malaria susceptibility, it allowed us to explore and understand the intricate host–parasite interactions during malaria infections. In this study, we described a gene encoding erythrocytic ankyrin-1 (Ank-1) which exhibits allelic-dependent heterogeneous phenotypes during malaria infections. We conducted an ENU mutagenesis screen on mice and identified two Ank-1 mutations, one resulting in an amino acid substitution (MRI95845), and the other a truncated Ank-1 protein (MRI96570). Both mutations caused hereditary spherocytosis-like phenotypes and confer differing protection against Plasmodium chabaudi infections. Upon further examination, the Ank-1(MRI96570) mutation was found to inhibit intraerythrocytic parasite maturation, whereas Ank-1(MRI95845) caused increased bystander erythrocyte clearance during infection. This is the first description of allelic heterogeneity in ankyrin-1 from the direct comparison between two Ank-1 mutations. Despite the lack of direct evidence from population studies, this data further supported the protective roles of ankyrin-1 mutations in conferring malaria protection. This study also emphasized the importance of such phenomena in achieving a better understanding of host–parasite interactions, which could be the basis of future studies.
Publisher: Public Library of Science (PLoS)
Date: 13-06-2017
Publisher: Wiley
Date: 10-2009
DOI: 10.1111/J.1558-5646.2009.00737.X
Abstract: To assess the genetic basis of the skull shape variation and morphological integration in mice, we have used a tool based on the cross between the distantly related mouse species Mus spretus (SEG/Pas strain) and the laboratory strain C57BL/6 called interspecific recombinant congenic strains (IRCSs). The genome of each IRCS consists on average of 1.3% of SEG/Pas derived sequences, located on multiple chromosomes as small-sized, DNA segments. Quantitative trait loci (QTL) on the skull shape, separated into dorsal and ventral sides, were analyzed in 17 IRCSs by a Procrustes superimposition method using 3D landmarks. The shapes of 16 strains differed significantly from C57BL/6. Discrepancy in the QTLs effects was found between the dorsal side and the anterior region of the ventral side due to a differential effect of the SEG/Pas alleles on the skull shape. A comprehensive analysis of all allelic combinations of the BCG-66H strain showed strong epistatic interactions between SEG/Pas segment acting on both skull sides. Epistatic pleiotropy and covariation between sides were dependent in SEG/Pas alleles direction and contributed to the strong morphological integration between sides. Introduction of Mus spretus alleles in a C57BL/6 background induced strong morphological changes mostly in SEG/Pas alleles direction and provided evidence for high level of morphological integration.
Publisher: Springer Science and Business Media LLC
Date: 28-03-2018
DOI: 10.1007/S00335-018-9744-9
Abstract: Malaria remains a deadly parasitic disease caused by Plasmodium, claiming almost half a million lives every year. While parasite genetics and biology are often the major targets in many studies, it is becoming more evident that host genetics plays a crucial role in the outcome of the infection. Similarly, Plasmodium infections in mice also rely heavily on the genetic background of the mice, and often correlate with observations in human studies, due to their high genetic homology with humans. As such, murine models of malaria are a useful tool for understanding host responses during Plasmodium infections, as well as dissecting host-parasite interactions through various genetic manipulation techniques. Reverse genetic approach such as quantitative trait loci studies and random mutagenesis screens have been employed to discover novel host genes that affect malaria susceptibility in mouse models, while other targeted studies utilize mouse models to validate observation from human studies. Herein, we review the findings from the past and present studies on murine models of hepatic and erythrocytic stages of malaria and speculate on how the current mouse models benefit from the recent development in CRISPR/Cas9 gene editing technology.
Publisher: The Royal Society
Date: 10-2016
DOI: 10.1098/RSOB.160109
Abstract: Recurrent spontaneous abortion (RSA) is a common cause of infertility, but previous attempts at identifying RSA causative genes have been relatively unsuccessful. Such failure to describe RSA aetiological genes might be explained by the fact that reproductive phenotypes should be considered as quantitative traits resulting from the intricate interaction of numerous genetic, epigenetic and environmental factors. Here, we studied an interspecific recombinant congenic strain (IRCS) of Mus musculus from the C57BL6/J strain of mice harbouring an approximate 5 Mb DNA fragment from chromosome 13 from Mus spretus mice (66H-MMU13 strain), with a high rate of embryonic resorption (ER). Transcriptome analyses of endometrial and placental tissues from these mice showed a deregulation of many genes associated with the coagulation and inflammatory response pathways. Bioinformatics approaches led us to select Foxd1 as a candidate gene potentially related to ER and RSA. Sequencing analysis of Foxd1 in the 66H-MMU13 strain, and in 556 women affected by RSA and 271 controls revealed non-synonymous sequence variants. In vitro assays revealed that some led to perturbations in FOXD1 transactivation properties on promoters of genes having key roles during implantation lacentation, suggesting a role of this gene in mammalian implantation processes.
Publisher: Springer Science and Business Media LLC
Date: 26-08-2019
DOI: 10.1186/S13059-019-1776-2
Abstract: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.
Publisher: American Society of Hematology
Date: 12-12-2017
DOI: 10.1182/BLOODADVANCES.2017009274
Abstract: Mutations in β spectrin cause microcytosis, resulting in increased clearance of erythrocytes and enhanced resistance to malaria in mice. A homozygous CRISPR/Cas9-induced mutation in the binding site between β spectrin and ankyrin-1 increases mouse survival during malaria.
Publisher: Springer Science and Business Media LLC
Date: 17-03-2014
Publisher: Springer Science and Business Media LLC
Date: 05-08-2019
DOI: 10.1007/S40259-019-00369-Y
Abstract: The class 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, one of the prokaryotic adaptive immune systems, has sparked a lot of attention for its use as a gene editing tool. Currently, type II, V, and VI effector modules of this class have been characterized and extensively tested for nucleic acid editing, imaging, and disease diagnostics. Due to the unique composition of their nuclease catalytic center, the effector modules substantially vary in their function and possible biotechnology applications. In this review, we discuss the structural and functional ersity in class 2 CRISPR effectors, and debate their suitability for nucleic acid targeting and their shortcomings as gene editing tools.
Publisher: Frontiers Media SA
Date: 16-04-2019
Publisher: Springer Science and Business Media LLC
Date: 19-12-2017
DOI: 10.1038/S41598-017-17861-6
Abstract: Glutathione transferase Omega 1 (GSTO1-1) is an atypical GST reported to play a pro-inflammatory role in response to LPS. Here we show that genetic knockout of Gsto1 alters the response of mice to three distinct inflammatory disease models. GSTO1-1 deficiency ameliorates the inflammatory response stimulated by LPS and attenuates the inflammatory impact of a high fat diet on glucose tolerance and insulin resistance. In contrast, GSTO1-1 deficient mice show a more severe inflammatory response and increased escape of bacteria from the colon into the lymphatic system in a dextran sodium sulfate mediated model of inflammatory bowel disease. These responses are similar to those of TLR4 and MyD88 deficient mice in these models and confirm that GSTO1-1 is critical for a TLR4-like pro-inflammatory response in vivo . In wild-type mice, we show that a small molecule inhibitor that covalently binds in the active site of GSTO1-1 can be used to ameliorate the inflammatory response to LPS. Our findings demonstrate the potential therapeutic utility of GSTO1-1 inhibitors in the modulation of inflammation and suggest their possible application in the treatment of a range of inflammatory conditions.
Publisher: Public Library of Science (PLoS)
Date: 19-06-2012
Publisher: Cold Spring Harbor Laboratory
Date: 30-08-2018
DOI: 10.1101/393231
Abstract: CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles ( %), and a very small number of desired alleles ( %) requiring, on average 1,192 zygotes.
Publisher: Oxford University Press (OUP)
Date: 15-09-2022
DOI: 10.1093/NAR/GKAC767
Abstract: Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.
Publisher: EMBO
Date: 10-02-2023
Abstract: Moraxella catarrhalis is an important human respiratory pathogen and a major causative agent of otitis media and chronic obstructive pulmonary disease. Toll‐like receptors contribute to, but cannot fully account for, the complexity of the immune response seen in M. catarrhalis infection. Using primary mouse bone marrow‐derived macrophages to examine the host response to M. catarrhalis infection, our global transcriptomic and targeted cytokine analyses revealed activation of immune signalling pathways by both membrane‐bound and cytosolic pattern‐recognition receptors. We show that M. catarrhalis and its outer membrane vesicles or lipooligosaccharide (LOS) can activate the cytosolic innate immune sensor caspase‐4/11, gasdermin‐D‐dependent pyroptosis, and the NLRP3 inflammasome in human and mouse macrophages. This pathway is initiated by type I interferon signalling and guanylate‐binding proteins (GBPs). We also show that inflammasomes and GBPs, particularly GBP2, are required for the host defence against M. catarrhalis in mice. Overall, our results reveal an essential role for the interferon‐inflammasome axis in cytosolic recognition and immunity against M. catarrhalis , providing new molecular targets that may be used to mitigate pathological inflammation triggered by this pathogen.
Start Date: 2013
End Date: 2016
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2016
End Date: 2019
Funder: Marsden Fund
View Funded ActivityStart Date: 2014
End Date: 2016
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2010
End Date: 2012
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 07-2018
End Date: 12-2021
Amount: $487,460.00
Funder: Australian Research Council
View Funded Activity