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Item Metadata only 3D printed lattices as an activation and expansion platform for T cell therapy(Elsevier, 2017) Delalat, B.; Harding, F.; Gundsambuu, B.; De-Juan-Pardo, E.; Wunner, F.; Wille, M.; Jasieniak, M.; Malatesta, K.; Griesser, H.; Simula, A.; Hutmacher, D.; Voelcker, N.; Barry, S.Abstract not availableItem Metadata only A case of haemorrhagic myositis with concurrent anti-Ro52 and anti-NXP-2 antibodies treated with plasmapheresis(Oxford University Press, 2020) Brown, Z.R.; Thomas, J.S.; Limaye, V.Abstract not availableItem Open Access A cell permeable bimane-constrained PCNA-interacting peptide(Royal Society of Chemistry (RSC), 2021) Horsfall, A.J.; Vandborg, B.; Kikhtyak, Z.; Scanlon, D.B.; Tilley, W.D.; Hickey, T.E.; Bruning, J.B.; Abell, A.D.The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity via a 3₁₀-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143–151) conformationally constrained with a covalent linker between i, i + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with K(D) values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide 7 proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide 7 adopted the classical 3₁₀-helical binding conformation. This suggests the 3₁₀-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide 7 revealed this secondary structure was not well-defined in solution. Peptide 7 is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.Item Metadata only A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Megalania) priscus(Natl Acad Sciences, 2009) Scanlon, D.The predatory ecology of Varanus komodoensis (Komodo Dragon)has been a subject of long-standing interest and considerable conjecture. Here, we investigate the roles and potential interplay between cranial mechanics, toxic bacteria, and venom. Our analyses point to the presence of a sophisticated combined-arsenal killing apparatus. We find that the lightweight skull is relatively poorly adapted to generate high bite forces but better adapted to resist high pulling loads. We reject the popular notion regarding toxic bacteria utilization. Instead, we demonstrate that the effects of deep wounds inflicted are potentiated through venom with toxic activities including anticoagulation and shock induction. Anatomical comparisons of V. komodoensis with V.(Megalania) priscus fossils suggest that the closely related extinct giant was the largest venomous animal to have ever lived.Item Metadata only A composite fibre optic catheter for monitoring peristaltic transit of an intra-luminal bead(Wiley-VCH, 2016) Arkwright, J.; Underhill, I.; Dodds, K.; Brookes, S.; Costa, M.; Spencer, N.; Dinning, P.Cut-away image of the fibre optic motion sensor showing the location of the fibre Bragg gratings and the rare earth magnet. A fibre optic motion sensor has been developed for monitoring the proximity and direction of motion of a ferrous bead travelling axial to the sensor. By integrating an array of these sensors into our previously developed fibre optic manometry catheters we demonstrate simultaneous detection of peristaltic muscular activity and the associated motion of ferrous beads through a colonic lumen. This allows the motion of solid content to be temporally and spatially related to pressure variations generated by peristaltic contractions without resorting to videoflouroscopy to track the motion of a radio opaque bolus. The composite catheter has been tested in an in-vitro animal preparation consisting of excised sections of rabbit colon.Item Metadata only A covalently dimerized recombinant human bone morphogenetic protein-15 variant identifies bone morphogenetic protein receptor type 1B as a key cell surface receptor on ovarian granulosa cells(Endocrine Soc, 2012) Pulkki, M.; Mottershead, D.; Pasternack, A.; Muggalla, P.; Ludlow, H.; van Dinther, M.; Myllymaa, S.; Koli, K.; ten Dijke, P.; Laitinen, M.; Ritvos, O.Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [125I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [125I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [125I]BMP15S356C variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [125I]BMP15S356C variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant.Item Open Access A CX3CR1 reporter hESC line facilitates integrative analysis of in-vitro-derived microglia and improved microglia identity upon neuron-glia co-culture(Elsevier, 2020) Grubman, A.; Vandekolk, T.H.; Schröder, J.; Sun, G.; Hatwell-Humble, J.; Chan, J.; Oksanen, M.; Lehtonen, S.; Hunt, C.; Koistinaho, J.E.; Nilsson, S.K.; Haynes, J.M.; Pouton, C.W.; Polo, J.M.Multiple protocols have been published for generation of iMGLs from hESCs/iPSCs. To date, there are no guides to assist researchers to determine the most appropriate methodology for microglial studies. To establish a framework to facilitate future microglial studies, we first performed a comparative transcriptional analysis between iMGLs derived using three published datasets, which allowed us to establish the baseline protocol that is most representative of bona fide human microglia. Secondly, using CRISPR to tag the classic microglial marker CX3CR1 with nanoluciferase and tdTomato, we generated and functionally validated a reporter ESC line. Finally, using this cell line, we demonstrated that co-culture of iMGL precursors with human glia and neurons enhanced transcriptional resemblance of iMGLs to ex vivo microglia. Together, our comprehensive molecular analysis and reporter cell line are a useful resource for neurobiologists seeking to use iMGLs for disease modeling and drug screening studies.Item Metadata only A double-blind randomized controlled trial of ibuprofen compared to placebo for uncomplicated cellulitis of the upper or lower limb(Elsevier, 2017) Davis, J.S.; Mackrow, C.; Binks, P.; Fletcher, W.; Dettwiller, P.; Marshall, C.; Day, J.; Pratt, W.; Tong, S.Y.C.Objectives: Cellulitis is a common skin infection resulting in inflammation that may take weeks to resolve despite appropriate antibiotics. It is unclear whether the adjunctive use of nonsteroidal anti-inflammatory drugs hastens the resolution of inflammation in patients with cellulitis. Methods: We conducted a double-blind, randomized controlled trial comparing ibuprofen 400 mg three times daily for 5 days with identical placebo in adults with uncomplicated cellulitis of the upper or lower limb who were treated with intravenous cefazolin via an outpatient parenteral antibiotic treatment service at one of two Australian hospitals. Participants were assessed twice daily by a study nurse. The primary outcome measure was the proportion of patients with regression of inflammation 48 hours after the first effective dose of parenteral antibiotics (trial registration ANZCTR 12611000515998). Results: Fifty-one patients were enrolled; 48 had sufficient data available to be included in the modified intention-to-treat analysis. Inflammation had begun to regress at 48 hours in 20 participants (80%) in the ibuprofen group compared to 15 (65%) in the placebo group (absolute risk difference +15%; 95% confidence interval −10 to +40; p >0.05). There was no significant difference in any secondary outcome. Ibuprofen appeared safe, with no patients developing renal impairment or necrotizing fasciitis. Conclusions: This trial demonstrated no significant benefit of adjunctive ibuprofen in adults with uncomplicated cellulitis. The trial was powered to detect a large effect, and hence it is unclear whether the 15% absolute increase in the primary end point in the ibuprofen group was attributable to chance. Previous article in issueItem Metadata only A history of carpal tunnel syndrome(Mark Allen Publishing Ltd, 2008) Wardle, N.; Pourgiezis, N.; Ashwood, N.; Bain, G.Carpal tunnel syndrome is the commonest entrapment neuropathy seen in clinical practice. The history of its aetiology and diagnosis gives an interesting insight into how the condition has evolved to also become the best understood neuropathy.Item Open Access A hybrid mechanism of action for BCL6 in B cells defined by formation of functionally distinct complexes at enhancers and promoters(Cell Press, 2013) Hatzi, K.; Jiang, Y.; Huang, C.; Garrett-Bakelman, F.; Gearhart, M.D.; Giannopoulou, E.G.; Zumbo, P.; Kirouac, K.; Bhaskara, S.; Polo, J.M.; Kormaksson, M.; MacKerell, A.D.; Xue, F.; Mason, C.E.; Hiebert, S.W.; Prive, G.G.; Cerchietti, L.; Bardwell, V.J.; Elemento, O.; Melnick, A.The BCL6 transcriptional repressor is required for the development of germinal center (GC) B cells and diffuse large B cell lymphomas (DLBCLs). Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B cells is unknown. We find that in B cells, BCL6 mostly functions through two independent mechanisms that are collectively essential to GC formation and DLBCL, both mediated through its N-terminal BTB domain. These are (1) the formation of a unique ternary BCOR-SMRT complex at promoters, with each corepressor binding to symmetrical sites on BCL6 homodimers linked to specific epigenetic chromatin features, and (2) the "toggling" of active enhancers to a poised but not erased conformation through SMRT-dependent H3K27 deacetylation, which is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Dynamic toggling of enhancers provides a basis for B cells to undergo rapid transcriptional and phenotypic changes in response to signaling or environmental cues.Item Metadata only A lineage of diploid platelet-forming cells precedes polyploid megakaryocyte formation in the mouse embryo(American Society of Hematology, 2014) Potts, K.S.; Sargeant, T.J.; Markham, J.F.; Shi, W.; Biben, C.; Josefsson, E.C.; Whitehead, L.W.; Rogers, K.L.; Liakhovitskaia, A.; Smyth, G.K.; Kile, B.T.; Medvinsky, A.; Alexander, W.S.; Hilton, D.J.; Taoudi, S.In this study, we test the assumption that the hematopoietic progenitor/colony-forming cells of the embryonic yolk sac (YS), which are endowed with megakaryocytic potential, differentiate into the first platelet-forming cells in vivo. We demonstrate that from embryonic day (E) 8.5 all megakaryocyte (MK) colony-forming cells belong to the conventional hematopoietic progenitor cell (HPC) compartment. Although these cells are indeed capable of generating polyploid MKs, they are not the source of the first platelet-forming cells. We show that proplatelet formation first occurs in a unique and previously unrecognized lineage of diploid platelet-forming cells, which develop within the YS in parallel to HPCs but can be specified in the E8.5 Runx1-null embryo despite the absence of the progenitor cell lineage.Item Open Access A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs(Cold Spring Harbor Laboratory, 2018) Pflueger, C.; Tan, D.; Swain, T.; Nguyen, T.; Pflueger, J.; Nefzger, C.; Polo, J.M.; Ford, E.; Lister, R.Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.Item Metadata only A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen(Company of Biologists, 2017) Conway, A.J.; Brown, F.C.; Fullinfaw, R.O.; Kile, B.T.; Jane, S.M.; Curtis, D.J.A genome-wide ethyl-N-nitrosourea (ENU) mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX). A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.Item Metadata only A move to more systematic and transparent approaches in qualitative evidence synthesis: update on a review of published papers(Sage Publications, 2012) Hannes, K.; Macaitis, K.In 2007, the journal Qualitative Research published a review on qualitative evidence syntheses conducted between 1988 and 2004. It reported on the lack of explicit detail regarding methods for searching, appraisal and synthesis, and a lack of emerging consensus on these issues. We present an update of this review for the period 2005–8. Not only has the amount of published qualitative evidence syntheses doubled, but authors have also become more transparent about their searching and critical appraisal procedures. Nevertheless, for the synthesis component of the qualitative reviews, a black box remains between what people claim to use as a synthesis approach and what is actually done in practice. A detailed evaluation of how well authors master their chosen approach could provide important information for developers of particular methods, who seem to succeed in playing the game according to the rules. Clear methodological instructions need to be developed to assist others in applying these synthesis methods.Item Metadata only A new bifunctional chelator for copper radiopharmaceuticals: a cage amine ligand with a carboxylate functional group for conjugation to peptides(Royal Soc Chemistry, 2009) Ma, M.; Karas, J.; White, J.; Scanlon, D.; Donnelly, P.A new sarcophagine cage amine ligand with a pendent carboxylate functional group has been synthesised; the ligand has been conjugated to tumour targeting peptides ([Tyr3]- octreotate and [Lys3]-bombesin) and the conjugates radiolabelled with copper-64.Item Open Access A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.(Springer Science and Business Media LLC, 2023) Mayoh, C.; Gifford, A.J.; Terry, R.; Lau, L.M.S.; Wong, M.; Rao, P.; Shai-Hee, T.; Saletta, F.; Khuong-Quang, D.-A.; Qin, V.; Mateos, M.K.; Meyran, D.; Miller, K.E.; Yuksel, A.; Mould, E.V.A.; Bowen-James, R.; Govender, D.; Senapati, A.; Zhukova, N.; Omer, N.; et al.BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.Item Metadata only A possible cause of Alzheimer's dementia - industrial soy foods(Elsevier, 2014) Roccisano, D.; Henneberg, M.; Saniotis, A.Abstract not availableItem Metadata only A primer extension denaturing high-performance liquid chromatography method for the identification of three ABCC2 genetic polymorphisms(Taylor and Francis, 2014) Westley, I.; Coller, J.; Ward, M.; Evans, A.; Morris, R.; Sallustio, B.Background: A number of single nucleotide polymorphisms have been described in the ABCC2 gene that alters drug disposition. The aim of the study was to develop a primer extension denaturing high-performance liquid chromatography (PE-dHPLC) assay to determine three common variants of the ABCC2 gene in the promoter region (−24C>T), exon 10 (1249G>A), and exon 28 (3972C>T). Methods: Polymerase chain reactions (PCRs) were used to isolate the area of interest in the ABCC2 gene. A comparison of the PCR product was performed between sequencing and dHPLC. Results: A 100% identity match was achieved between groups and allowed for a quick and accurate method to determine three single nucleotide polymorphisms in a single extension reaction. This assay is the first of its type to determine three ABCC2 variants by dHPLCItem Metadata only A proteomic approach to identifying matrix proteins interacting with transforming growth factor-β-inducible gene h-3 (βig-h3)(Matrix Biology Society of Australia and New Zealand, 2005) Gibson, M.; Kamkar Parsi, M.; Hanssen, E.; Annual Meeting of the Matrix Biology Society of Australia and New Zealand (29th : 2005 : Victor Harbor, S. Aust.)Item Open Access A randomised controlled trial of losartan as an anti-fibrotic agent in non-alcoholic steatohepatitis(Public Library Science, 2017) McPherson, S.; Wilkinson, N.; Tiniakos, D.; Wilkinson, J.; Burt, A.; McColl, E.; Stocken, D.; Steen, N.; Barnes, J.; Goudie, N.; Stewart, S.; Bury, Y.; Mann, D.; Anstee, Q.; Day, C.; Wong, V.Non-alcoholic fatty liver disease (NAFLD) is a common liver disease worldwide. Experimental and small clinical trials have demonstrated that angiotensin II blockers (ARB) may be anti-fibrotic in the liver. The aim of this randomised controlled trial was to assess whether treatment with Losartan for 96 weeks slowed, halted or reversed the progression of fibrosis in patients with non-alcoholic steatohepatitis (NASH).Double-blind randomised-controlled trial of Losartan 50 mg once a day versus placebo for 96 weeks in patients with histological evidence of NASH. The primary outcome for the study was change in histological fibrosis stage from pre-treatment to end-of-treatment.The study planned to recruit 214 patients. However, recruitment was slower than expected, and after 45 patients were randomised (median age 55; 56% male; 60% diabetic; median fibrosis stage 2), enrolment was suspended. Thirty-two patients (15 losartan and 17 placebo) completed follow up period: one patient (6.7%) treated with losartan and 4 patients (23.5%) in the placebo group were "responders" (lower fibrosis stage at follow up compared with baseline). The major reason for slow recruitment was that 39% of potentially eligible patients were already taking an ARB or angiotensin converting enzyme inhibitor (ACEI), and 15% were taking other prohibited medications.Due to the widespread use of ACEI and ARB in patients with NASH this trial failed to recruit sufficient patients to determine whether losartan has anti-fibrotic effects in the liver.ISRCTN 57849521.