School of Biomedical Sciences - Research Publications
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ItemHost-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter(NATURE PORTFOLIO, 2022-11-30)Described antimicrobial resistance mechanisms enable bacteria to avoid the direct effects of antibiotics and can be monitored by in vitro susceptibility testing and genetic methods. Here we describe a mechanism of sulfamethoxazole resistance that requires a host metabolite for activity. Using a combination of in vitro evolution and metabolic rescue experiments, we identify an energy-coupling factor (ECF) transporter S component gene (thfT) that enables Group A Streptococcus to acquire extracellular reduced folate compounds. ThfT likely expands the substrate specificity of an endogenous ECF transporter to acquire reduced folate compounds directly from the host, thereby bypassing the inhibition of folate biosynthesis by sulfamethoxazole. As such, ThfT is a functional equivalent of eukaryotic folate uptake pathways that confers very high levels of resistance to sulfamethoxazole, yet remains undetectable when Group A Streptococcus is grown in the absence of reduced folates. Our study highlights the need to understand how antibiotic susceptibility of pathogens might function during infections to identify additional mechanisms of resistance and reduce ineffective antibiotic use and treatment failures, which in turn further contribute to the spread of antimicrobial resistance genes amongst bacterial pathogens.
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ItemInsect Antennal Morphology: The Evolution of Diverse Solutions to Odorant Perception(Yale University, 2018-12-01)Chemical communication involves the production, transmission, and perception of odors. Most adult insects rely on chemical signals and cues to locate food resources, oviposition sites or reproductive partners and, consequently, numerous odors provide a vital source of information. Insects detect these odors with receptors mostly located on the antennae, and the diverse shapes and sizes of these antennae (and sensilla) are both astonishing and puzzling: what selective pressures are responsible for these different solutions to the same problem - to perceive signals and cues? This review describes the selection pressures derived from chemical communication that are responsible for shaping the diversity of insect antennal morphology. In particular, we highlight new technologies and techniques that offer exciting opportunities for addressing this surprisingly neglected and yet crucial component of chemical communication.
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ItemThe effect of substance P and its common in vivo-formed metabolites on MRGPRX2 and human mast cell activation(JOHN WILEY & SONS LTD, 2022-08)The tachykinin neuropeptide substance P (SP) is the canonical agonist peptide for the neurokinin 1 receptor (NK1 R). More recently, it has also been shown to activate the Mas-related G protein-coupled receptor X2 (MRGPRX2) receptor on mast cells (MCs), triggering degranulation and release of inflammatory mediators. SP undergoes rapid C-terminal truncation in vivo by a number of proteases to generate the metabolites SP(1-9)-COOH and in particular SP(1-7)-COOH. While the C terminus of SP is critical for NK1 R activation, studies have shown that the peptide polycationic N terminus is key for MRGPRX2 and mast cell activation. The study thus aimed to determine if the C-terminally truncated metabolites of SP, SP(1-9)-COOH, and SP(1-7)-COOH retained stimulatory activity at MRGPRX2. SP, SP(1-9)-COOH, and SP(1-7)-COOH were synthesized and tested on HEK293 cells expressing NK1 R or MRGPRX2, and LAD2 human mast cells, to determine the activity of SP and its metabolites in Ca2+ mobilization, degranulation, and cytokine assays. As expected from prior studies, both C-terminally truncated SP metabolites had essentially no activity at NK1 R, even at very high concentrations. In contrast, the in vivo metabolite of SP, SP(1-9)-COOH retained ability to activate MRGPRX2 across all parameters tested, albeit with reduced potency compared to intact SP. SP(1-7)-COOH did not produce any significant MRGRPX2 activation. Our results suggest that the SP metabolite, SP(1-9)-COOH, may play a regulatory role through the activation of MRGPRX2. However, given the relatively low potency of both SP and SP(1-9)-COOH at MRGPRX2, additional work is needed to better understand the biological importance of this expanded SP/MRGPRX2 pathway.
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ItemTetraspanin CD82 restrains phagocyte migration but supports macrophage activation(CELL PRESS, 2022-07-15)Phagocytes migrate into tissues to combat infection and maintain tissue homeostasis. As dysregulated phagocyte migration and function can lead to inflammation or susceptibility to infection, identifying molecules that control these processes is critical. Here, we show that the tetraspanin CD82 restrains the migration of neutrophils and macrophages into tissues. Cd82 -/- phagocytes exhibited excessive migration during in vivo models of peritoneal inflammation, superfusion of CXCL1, retinopathy of prematurity, and infection with the protozoan parasite L. mexicana. However, with the latter, while Cd82 -/- macrophages infiltrated infection sites at higher proportions, cutaneous L. mexicana lesions were larger and persisted, indicating a failure to control infection. Analyses of in vitro bone-marrow-derived macrophages showed CD82 deficiency altered cellular morphology, and impaired gene expression and metabolism in response to anti-inflammatory activation. Altogether, this work reveals an important role for CD82 in restraining phagocyte infiltration and mediating their differentiation in response to stimulatory cues.
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ItemThe potentially beneficial central nervous system activity profile of ivacaftor and its metabolites (vol 4, 00127, 2018)(EUROPEAN RESPIRATORY SOC JOURNALS LTD, 2018-10-01)[This corrects the article DOI: 10.1183/23120541.00127-2017.].
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ItemCharge Has a Marked Influence on Hyperbranched Polymer Nanoparticle Association in Whole Human Blood(AMER CHEMICAL SOC, 2017-06)In this study, we synthesize charge-varied hyperbranched polymers (HBPs) and demonstrate surface charge as a key parameter directing their association with specific human blood cell types. Using fresh human blood, we investigate the association of 5 nm HBPs with six white blood cell populations in their natural milieu by flow cytometry. While most cell types associate with cationic HBPs at 4 °C, at 37 °C phagocytic cells display similar (monocyte, dendritic cell) or greater (granulocyte) association with anionic HBPs compared to cationic HBPs. Neutral HBPs display remarkable stealth properties. Notably, these charge-association patterns are not solely defined by the plasma protein corona and are material and/or size dependent. As HBPs progress toward clinical use as imaging and drug delivery agents, the ability to engineer HBPs with defined biological properties is increasingly important. This knowledge can be used in the rational design of HBPs for more effective delivery to desired cell targets.
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ItemHypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor-A expression and downregulation of miRNA-1, miRNA-206 and angiopoietin-1(WILEY, 2018-01)Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR-1 and miR-206 (p < 0.05) and angiopoietin-1 (p < 0.05) with upregulation of vascular endothelial growth factor-A (VEGF-A; p < 0.05). The miR-1 and angiopoietin-1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR-206 in L6 myoblasts caused a significant increase in VEGF-A expression (p < 0.05), further establishing that changes in VEGF-A expression are influenced by miR-206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel™ into isolated bilateral tissue engineering chambers incorporating a flow-through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin-1, miR-1 and miR-206. The relatively simple strategy of hypoxic preconditioning of implanted cells - including non-stem cell types - has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.
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ItemObservational study of alternative therapies among paediatric emergency department patients(WILEY, 2017-04)OBJECTIVE: While complementary medicine use among ED paediatric patients is common, the use of alternative therapies (ATs; physical or spiritual therapies) is unknown. We aimed to determine the 12 month period prevalence and nature of AT use among paediatric patients and parent perceptions of AT use. METHODS: We undertook a cross-sectional survey of a convenience sample of parents of paediatric patients in three EDs in metropolitan Melbourne, Australia (January-June, 2015). Parents were invited to complete a validated, anonymous, self-administered questionnaire. The main outcomes were AT use by the patient and parent perceptions of ATs. RESULTS: A total of 806 parents were enrolled. In the previous 12 months, 393 (48.8%) patients had received at least one AT. There were no gender or ethnicity differences between AT users and non-users. AT use was more common among older patients (P < 0.05). Patients with chronic illness tended to use more ATs (P = 0.12). A total of 1091 courses of 43 different ATs had been provided. The most common were massage (16% of patients), chiropractic therapy (9.8%), relaxation (7.2%), meditation (6.2%) and aromatherapy (6.1%). ATs were generally used for musculoskeletal problems, health maintenance, stress and anxiety. Parents who arranged the ATs were significantly more likely to report that ATs are safe, prevent and treat illness, assist prescription medicines and offer a more holistic approach to healthcare (P < 0.001). CONCLUSION: AT use is common among paediatric ED patients. Parents who arrange AT have differing perceptions of AT usefulness and safety from those who do not.
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ItemCell cycle-regulated PLEIADE/AtMAP65-3 links membrane and microtubule dynamics during plant cytokinesis(WILEY-BLACKWELL, 2016-11)Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between cell cycle cues, membrane trafficking and microtubule dynamics. Plant cytokinesis occurs within a transient membrane compartment known as the cell plate, to which vesicles are delivered by a plant-specific microtubule array, the phragmoplast. While membrane proteins required for cytokinesis are known, how these are coordinated with microtubule dynamics and regulated by cell cycle cues remains unclear. Here, we document physical and genetic interactions between Transport Protein Particle II (TRAPPII) tethering factors and microtubule-associated proteins of the PLEIADE/AtMAP65 family. These interactions do not specifically affect the recruitment of either TRAPPII or MAP65 proteins to the cell plate or midzone. Rather, and based on single versus double mutant phenotypes, it appears that they are required to coordinate cytokinesis with the nuclear division cycle. As MAP65 family members are known to be targets of cell cycle-regulated kinases, our results provide a conceptual framework for how membrane and microtubule dynamics may be coordinated with each other and with the nuclear cycle during plant cytokinesis.
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ItemEffect of NADPH oxidase 1 and 4 blockade in activated human retinal endothelial cells(WILEY, 2018-08)BACKGROUND: Over-production of reactive oxygen species (ROS) and resulting oxidative stress contribute to retinal damage in vascular diseases that include diabetic retinopathy, retinopathy of prematurity and major retinal vessel occlusions. NADPH oxidase (Nox) proteins are professional ROS-generating enzymes, and therapeutic targeting in these diseases has strong appeal. Pharmacological inhibition of Nox4 reduces the severity of experimental retinal vasculopathy. We investigated the potential application of this drug approach in humans. METHODS: Differential Nox enzyme expression was studied by real-time-quantitative polymerase chain reaction in primary human retinal endothelial cell isolates and a characterized human retinal endothelial cell line. Oxidative stress was triggered chemically in endothelial cells, by treatment with dimethyloxalylglycine (DMOG; 100 μM); Nox4 and vascular endothelial growth factor (VEGFA) transcript were measured; and production of ROS was detected by 2',7'-dichlorofluorescein. DMOG-stimulated endothelial cells were treated with two Nox1/Nox4 inhibitors, GKT136901 and GKT137831; cell growth was monitored by DNA quantification, in addition to VEGFA transcript and ROS production. RESULTS: Nox4 (isoform Nox4A) was the predominant Nox enzyme expressed by human retinal endothelial cells. Treatment with DMOG significantly increased endothelial cell expression of Nox4 over 72 h, accompanied by ROS production and increased VEGFA expression. Treatment with GKT136901 or GKT137831 significantly reduced DMOG-induced ROS production and VEGFA expression by endothelial cells, and the inhibitory effect of DMOG on cell growth. CONCLUSIONS: Our findings in experiments on activated human retinal endothelial cells provide translational corroboration of studies in experimental models of retinal vasculopathy and support the therapeutic application of Nox4 inhibition by GKT136901 and GKT137831 in patients with retinal vascular diseases.