Publications by authors named "Cameron J Nowell"

73 Publications

Mouse models for dominant dystrophic epidermolysis bullosa carrying common human point mutations recapitulate the human disease.

Dis Model Mech 2021 Jun 4;14(6). Epub 2021 Jun 4.

Murdoch Children's Research Institute, Parkville, VIC 3052, Australia.

Heterozygous missense mutations in the human COL7A1 gene - coding for collagen VII - lead to the rare, dominantly inherited skin disorder dominant dystrophic epidermolysis bullosa (DDEB), which is characterised by skin fragility, blistering, scarring and nail dystrophy. To better understand the pathophysiology of DDEB and develop more effective treatments, suitable mouse models for DDEB are required but to date none have existed. We identified the two most common COL7A1 mutations in DDEB patients (p.G2034R and p.G2043R) and used CRISPR-Cas9 to introduce the corresponding mutations into mouse Col7a1 (p.G2028R and p.G2037R). Dominant inheritance of either of these two alleles results in a phenotype that closely resembles that seen in DDEB patients. Specifically, mice carrying these alleles show recurrent blistering that is first observed transiently around the mouth and paws in the early neonatal period and then again around the digits from 5-10 weeks of age. Histologically, the mice show micro-blistering and reduced collagen VII immunostaining. Biochemically, collagen VII from these mice displays reduced thermal stability, which we also observed to be the case for DDEB patients carrying the analogous mutations. Unlike previous rodent models of epidermolysis bullosa, which frequently show early lethality and severe disease, these mouse models, which to our knowledge are the first for DDEB, show no reduction in growth and survival, and - together with a relatively mild phenotype - represent a practically and ethically tractable tool for better understanding and treating epidermolysis bullosa. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.048082DOI Listing
June 2021

High-content siRNA 3D co-cultures to identify myoepithelial cell-derived breast cancer suppressor proteins.

Sci Data 2021 05 28;8(1):147. Epub 2021 May 28.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, 3086, Australia.

Understanding how cancer cells interact with the surrounding microenvironment early in breast cancer development can provide insight into the initiation and progression of invasive breast cancers. The myoepithelial cell layer surrounding breast ducts acts as a physical barrier in early breast cancer, preventing cancer cells from invading the surrounding stroma. Changes to the expression profile and properties of myoepithelial cells have been implicated in progression to invasive carcinoma. Identifying the molecular drivers of myoepithelial cell-mediated tumour suppression may offer new approaches to predict and block the earliest stages of cancer invasion. We employed a high-content approach to knock down 87 different genes using siRNA in an immortalised myoepithelial cell line, prior to co-culture with invasive breast cancer cells in 3D. Combined with high-content imaging and a customised analysis pipeline, this system was used to identify myoepithelial proteins that are necessary to control cancer cell invasion. This dataset has identified prospective myoepithelial suppressors of early breast cancer invasion which may be used by researchers to investigate their clinical validity and utility.
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http://dx.doi.org/10.1038/s41597-021-00924-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163786PMC
May 2021

Physiological and pharmacological impact of oxytocin on epididymal propulsion during the ejaculatory process in rodents and men.

FASEB J 2021 Jun;35(6):e21639

Institute of Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany.

During the emission phase of ejaculation, the sperm is driven from the cauda epididymidis, where it is stored, through the vas deferens by strong contractions. These contractions are thought of as being mainly induced by the sympathetic nervous system and the neurotransmitter noradrenaline. In the present study, we investigated the effect of oxytocin (suggested to exert effects during ejaculation as well) on defined segments of the rat and human epididymis using live imaging. Our results indicate that it is the very last part of the epididymis, segment 19 (S19) in rat and likewise segment 9 in human, which responds in a uniquely strong and rapid manner to oxytocin (similar to noradrenaline). Because of the complex nature of this contractile response, we developed an imaging analysis method, which allowed us to quantify multidirectional contractions and to display them using heat maps. The reaction of S19 to oxytocin was concentration-dependent and could be inhibited by pretreatment with oxytocin antagonists (atosiban and cligosiban), but not with an arginine vasopressin 1 antagonist (SR49059). In both rat and human tissue, pretreatment with the alpha-1 adrenoreceptor antagonist tamsulosin inhibited the response to noradrenaline, whereas the effect of oxytocin was unimpaired. Our data (from men and rodents) strongly suggest that the hormone oxytocin is involved in the ejaculatory process. Thus, oxytocin-based medications might be a promising non-adrenergic treatment option for ejaculatory disorders. Additionally, we propose that S19 could be an advantageous model (detecting very low concentrations of oxytocin) to test the bioactivity of new oxytocin agonists and oxytocin antagonists.
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http://dx.doi.org/10.1096/fj.202100435RDOI Listing
June 2021

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

bioRxiv 2021 Mar 26. Epub 2021 Mar 26.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.
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http://dx.doi.org/10.1101/2021.03.22.436522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010724PMC
March 2021

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A Receptor in Living Cells.

J Med Chem 2021 05 16;64(10):6670-6695. Epub 2021 Mar 16.

Medicinal Chemistry, Monash University, Parkville, Victoria 3052, Australia.

The adenosine A receptor (AAR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of AAR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying AAR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of AAR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel AAR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of AAR molecular pharmacology and signaling in living cells.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02067DOI Listing
May 2021

The γH2AX DSB marker may not be a suitable biodosimeter to measure the biological MRT valley dose.

Int J Radiat Biol 2021 15;97(5):642-656. Epub 2021 Mar 15.

School of Science, RMIT University, Melbourne, Australia.

Purpose: γH2AX biodosimetry has been proposed as an alternative dosimetry method for microbeam radiation therapy (MRT) because conventional dosimeters, such as ionization chambers, lack the spatial resolution required to accurately measure the MRT valley dose. Here we investigated whether γH2AX biodosimetry should be used to measure the biological valley dose of MRT-irradiated mammalian cells.

Materials And Methods: We irradiated human skin fibroblasts and mouse skin flaps with synchrotron MRT and broad beam (BB) radiation. BB doses of 1-5 Gy were used to generate a calibration curve in order to estimate the biological MRT valley dose using the γH2AX assay.

Results: Our key finding was that MRT induced a non-linear dose response compared to BB, where doses 2-3 times greater showed the same level of DNA DSB damage in the valley in cell and tissue studies. This indicates that γH2AX may not be an appropriate biodosimeter to estimate the biological valley doses of MRT-irradiated samples. We also established foci yields of 5.9 ±  and 27.4 ±  foci/cell/Gy in mouse skin tissue and human fibroblasts respectively, induced by BB. Using Monte Carlo simulations, a linear dose response was seen in cell and tissue studies and produced predicted peak-to-valley dose ratios (PVDRs) of ∼30 and ∼107 for human fibroblasts and mouse skin tissue respectively.

Conclusions: Our report highlights novel MRT radiobiology, attempts to explain why γH2AX may not be an appropriate biodosimeter and suggests further studies aimed at revealing the biological and cellular communication mechanisms that drive the normal tissue sparing effect, which is characteristic of MRT.
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http://dx.doi.org/10.1080/09553002.2021.1893854DOI Listing
March 2021

Targeted delivery of mycophenolic acid to the mesenteric lymph node using a triglyceride mimetic prodrug approach enhances gut-specific immunomodulation in mice.

J Control Release 2021 Apr 17;332:636-651. Epub 2021 Feb 17.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria 3052, Australia. Electronic address:

The mesenteric lymph nodes (MLN) are a key site for the generation of adaptive immune responses to gut-derived antigenic material and immune cells within the MLN contribute to the pathophysiology of a range of conditions including inflammatory and autoimmune diseases, viral infections, graft versus host disease and cancer. Targeting immunomodulating drugs to the MLN may thus be beneficial in a range of conditions. This paper investigates the potential benefit of targeting a model immunosuppressant drug, mycophenolic acid (MPA), to T cells in the MLN, using a triglyceride (TG) mimetic prodrug approach. We confirmed that administration of MPA in the TG prodrug form (MPA-TG), increased lymphatic transport of MPA-related species 83-fold and increased MLN concentrations of MPA >20 fold, when compared to MPA alone, for up to 4 h in mice. At the same time, the plasma exposure of MPA and MPA-TG was similar, limiting the opportunity for systemic side effects. Confocal microscopy and flow cytometry studies with a fluorescent model prodrug (Bodipy-TG) revealed that the prodrug accumulated in the MLN cortex and paracortex at 5 and 10 h following administration and was highly associated with B cells and T cells that are found in these regions of the MLN. Finally, we demonstrated that MPA-TG was significantly more effective than MPA at inhibiting CD4 and CD8 T cell proliferation in the MLN of mice in response to an oral ovalbumin antigen challenge. In contrast, MPA-TG was no more effective than MPA at inhibiting T cell proliferation in peripheral LN when mice were challenged via SC administration of ovalbumin. This paper provides the first evidence of an in vivo pharmacodynamic benefit of targeting the MLN using a TG mimetic prodrug approach. The TG mimetic prodrug technology has the potential to benefit the treatment of a range of conditions where aberrant immune responses are initiated in gut-associated lymphoid tissues.
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http://dx.doi.org/10.1016/j.jconrel.2021.02.008DOI Listing
April 2021

Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

PLoS Pathog 2021 02 11;17(2):e1009165. Epub 2021 Feb 11.

Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.
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http://dx.doi.org/10.1371/journal.ppat.1009165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7877634PMC
February 2021

Depolymerization of hyaluronan using PEGylated human recombinant hyaluronidase promotes nanoparticle tumor penetration.

Nanomedicine (Lond) 2021 02 9;16(4):275-292. Epub 2021 Feb 9.

Drug Delivery, Disposition & Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.

Delivery of nanoparticles (NPs) to tumors can be impeded by high levels of hyaluronan (HA) in the stroma. Enzymatic depolymerization of HA with PEGylated hyaluronidase (PEGPH20) improves the delivery of antibodies to tumors. However, it is unknown whether NP delivery is enhanced by this strategy. The impact of PEGPH20 pretreatment on the uptake and tumor penetration of model PEGylated polystyrene NPs was studied in mice with orthotopic breast cancers. Tumor oxygenation and NP penetration, but not overall tumor uptake, of 50 nm NPs, was significantly enhanced by PEGPH20 pre-administration. PEGPH20 has the potential to improve intratumoral penetration of NP-based drug delivery systems and enhance access to cancer cells in poorly vascularized regions of the tumor.
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http://dx.doi.org/10.2217/nnm-2020-0433DOI Listing
February 2021

A lipid-anchored neurokinin 1 receptor antagonist prolongs pain relief by a three-pronged mechanism of action targeting the receptor at the plasma membrane and in endosomes.

J Biol Chem 2021 Jan 27:100345. Epub 2021 Jan 27.

Drug Discovery Biology Theme; Drug Delivery, Disposition and Dynamics Theme; Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology. Electronic address:

G protein-coupled receptors (GPCRs) are traditionally known for signaling at the plasma membrane, but they can also signal from endosomes after internalization to control important pathophysiological processes. In spinal neurons, sustained endosomal signaling of the neurokinin 1 receptor (NKR) mediates nociception, as demonstrated in models of acute and neuropathic pain. An NKR antagonist, Spantide I (Span), conjugated to cholestanol (Span-Chol), accumulates in endosomes, inhibits endosomal NKR signaling, and causes prolonged anti-nociception. However, the extent to which the Chol-anchor influences long-term location and activity is poorly understood. Herein, we used fluorescent correlation spectroscopy and targeted biosensors to characterize Span-Chol over time. The Chol-anchor increased local concentration of probe at the plasma membrane. Over time we observed an increase in NKR binding affinity and more potent inhibition of NKR-mediated calcium signaling. Span-Chol, but not Span, caused a persistent decrease in NKR recruitment of β-arrestin and receptor internalization to early endosomes. Using targeted biosensors, we mapped the relative inhibition of NKR signaling as the receptor moved into the cell. Span selectively inhibited cell surface signaling, whereas Span-Chol partitioned into endosomal membranes and blocked endosomal signaling. In a preclinical model of pain, Span-Chol caused prolonged antinociception (>9 h), which is attributable to a three-pronged mechanism of action: increased local concentration at membranes, a prolonged decrease in NKR endocytosis, and persistent inhibition of signaling from endosomes. Identifying the mechanisms that contribute to the increased preclinical efficacy of lipid-anchored NKR antagonists is an important step toward understanding how we can effectively target intracellular GPCRs in disease.
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http://dx.doi.org/10.1016/j.jbc.2021.100345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949131PMC
January 2021

No observed effect on brain vasculature of Alzheimer's disease-related mutations in the zebrafish presenilin 1 gene.

Mol Brain 2021 01 25;14(1):22. Epub 2021 Jan 25.

Alzheimer's Disease Genetics Laboratory, School of Biological Sciences, University of Adelaide, North Terrace, Adelaide, SA, 5005, Australia.

Previously, we found that brains of adult zebrafish heterozygous for Alzheimer's disease-related mutations in their presenilin 1 gene (psen1, orthologous to human PSEN1) show greater basal expression levels of hypoxia responsive genes relative to their wild type siblings under normoxia, suggesting hypoxic stress. In this study, we investigated whether this might be due to changes in brain vasculature. We generated and compared 3D reconstructions of GFP-labelled blood vessels of the zebrafish forebrain from heterozygous psen1 mutant zebrafish and their wild type siblings. We observed no statistically significant differences in vessel density, surface area, overall mean diameter, overall straightness, or total vessel length normalised to the volume of the telencephalon. Our findings do not support that changes in vascular morphology are responsible for the increased basal expression of hypoxia responsive genes in psen1 heterozygous mutant brains.
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http://dx.doi.org/10.1186/s13041-021-00734-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831246PMC
January 2021

Decreased Orexin Receptor 1 mRNA Expression in the Locus Coeruleus in Both Tau Transgenic rTg4510 and Tau Knockout Mice and Accompanying Ascending Arousal System Tau Invasion in rTg4510.

J Alzheimers Dis 2021 ;79(2):693-708

Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia.

Background: Sleep/wake disturbances (e.g., insomnia and sleep fragmentation) are common in neurodegenerative disorders, especially Alzheimer's disease (AD) and frontotemporal dementia (FTD). These symptoms are somewhat reminiscent of narcolepsy with cataplexy, caused by the loss of orexin-producing neurons. A bidirectional relationship between sleep disturbance and disease pathology suggests a detrimental cycle that accelerates disease progression and cognitive decline. The accumulation of brain tau fibrils is a core pathology of AD and FTD-tau and clinical evidence supports that tau may impair the orexin system in AD/FTD. This hypothesis was investigated using tau mutant mice.

Objective: To characterize orexin receptor mRNA expression in sleep/wake regulatory brain centers and quantify noradrenergic locus coeruleus (LC) and orexinergic lateral hypothalamus (LH) neurons, in tau transgenic rTg4510 and tau-/- mice.

Methods: We used i n situ hybridization and immunohistochemistry (IHC) in rTg4510 and tau-/- mice.

Results: rTg4510 and tau-/- mice exhibited a similar decrease in orexin receptor 1 (OX1R) mRNA expression in the LC compared with wildtype controls. IHC data indicated this was not due to decreased numbers of LC tyrosine hydroxylase-positive (TH) or orexin neurons and demonstrated that tau invades TH LC and orexinergic LH neurons in rTg4510 mice. In contrast, orexin receptor 2 (OX2R) mRNA levels were unaffected in either model.

Conclusion: The LC is strongly implicated in the regulation of sleep/wakefulness and expresses high levels of OX1R. These findings raise interesting questions regarding the effects of altered tau on the orexin system, specifically LC OX1Rs, and emphasize a potential mechanism which may help explain sleep/wake disturbances in AD and FTD.
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http://dx.doi.org/10.3233/JAD-201177DOI Listing
January 2021

Assessing the cellular toxicity of peptide inhibitors of intracellular protein-protein interactions by microinjection.

Bioorg Med Chem 2021 01 3;29:115906. Epub 2020 Dec 3.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic, Australia; ARC Centre for Fragment-Based Design, Monash University, Parkville, Victoria 3052, Australia. Electronic address:

Inhibitors of protein-protein interactions can be developed through a number of technologies to provide leads that include cell-impermeable molecules. Redesign of these impermeable leads to provide cell-permeable derivatives can be challenging and costly. We hypothesised that intracellular toxicity of leads could be assessed by microinjection prior to investing in the redesign process. We demonstrate this approach for our development of inhibitors of the protein-protein interaction between inducible nitric-oxide synthase (iNOS) and SPRY domain-containing SOCS box proteins (SPSBs). We microinjected a lead molecule into AD-293 cells and were able to perform an intracellular toxicity assessment. We also investigated the intracellular distribution and localisation of injected inhibitor using a fluorescently-labelled analogue. Our findings show that a lead peptide inhibitor, CP2, had no toxicity even at intracellular concentrations four orders of magnitude higher than its K for binding to SPSB2. This early toxicity assessment justifies further development of this cell-impermeable lead to confer cell permeability. Our investigation highlights the utility of microinjection as a tool for assessing toxicity during development of drugs targeting protein-protein interactions.
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http://dx.doi.org/10.1016/j.bmc.2020.115906DOI Listing
January 2021

Emulsions containing optimum cow milk fat and canola oil mixtures replicate the lipid self-assembly of human breast milk during digestion.

J Colloid Interface Sci 2021 Apr 24;588:680-691. Epub 2020 Nov 24.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, VIC 3052, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

Hypothesis: The digestion of different milks and milk substitutes leads to the formation of a variety of self-assembled lipid structures, with the structuring of human milk being paramount for infant nutrition. It was hypothesised that mixing cow milk fat rich in medium/long-chain lipids with canola oil rich in long-chain unsaturated lipids would replicate the structuring of human milk by balancing lipid chain lengths and saturation levels.

Experiments: Emulsions of cow milk fat/canola oil mixtures were prepared in two ways - by pre-mixing ghee and canola oil before dispersing them and by dispersing canola oil directly into commercial cow milk. Small angle X-ray scattering combined with titration of the fatty acids produced during digestion allowed for the correlation of dynamic lipid self-assembly with the extent of lipid digestion. Laser light scattering was used to show that the particle sizes in the digesting mixtures were similar and coherent anti-Stokes Raman spectroscopy (CARS) microscopy was used to confirm the mixing of canola oil into cow milk fat globules.

Findings: As the amount of long-chain unsaturated canola oil lipids in the mixtures increased, the lipid self-assembly tended towards colloidal structures of greater interfacial curvature. When the ratio of cow milk fat to canola oil lipids was 1:1 (w/w), the digesting lipids assembled themselves into the same liquid crystalline structures as human breast milk. This observation was independent of the method used to mix the lipids, with CARS microscopy indicating uniform mixing of the canola oil into cow milk upon ultrasonication.
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http://dx.doi.org/10.1016/j.jcis.2020.11.067DOI Listing
April 2021

Trisulfide linked cholesteryl PEG conjugate attenuates intracellular ROS and collagen-1 production in a breast cancer co-culture model.

Biomater Sci 2021 Feb;9(3):835-846

Australian Research Council - Centre of Excellence in Convergent Bio-Nano Science and Technology, Drug Delivery, Disposition and Dynamics Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia. and Department of Chemical Engineering, Faculty of Engineering, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.

The progression of cancer has been closely-linked with augmentation of cellular reactive oxygen species (ROS) levels and ROS-associated changes in the tumour microenvironment (TME), including alterations to the extracellular matrix and associated low drug uptake. Herein we report the application of a co-culture model to simulate the ROS based cell-cell interactions in the TME using fibroblasts and breast cancer cells, and describe how novel reactive polymers can be used to modulate those interactions. Under the co-culture conditions, both cell types exhibited modifications in behaviour, including significant overproduction of ROS in the cancer cells, and elevation of the collagen-1 secretion and stained actin filament intensity in the fibroblasts. To examine the potential of using reactive antioxidant polymers to intercept ROS communication and thereby manipulate the TME, we employed H2S-releasing macromolecular conjugates which have been previously demonstrated to mitigate ROS production in HEK cells. The specific conjugate used, mPEG-SSS-cholesteryl (T), significantly reduced ROS levels in co-cultured cancer cells by approximately 50%. This reduction was significantly greater than that observed with the other positive antioxidant controls. Exposure to T was also found to downregulate levels of collagen-1 in the co-cultured fibroblasts, while exhibiting less impact on cells in mono-culture. This would suggest a possible downstream effect of ROS-mitigation by T on stromal-tumour cell signalling. Since fibroblast-derived collagens modulate crucial steps in tumorigenesis, this ROS-associated effect could potentially be harnessed to slow cancer progression. The model may also be beneficial for interrogating the impact of antioxidants on naturally enhanced ROS levels, rather than relying on the application of exogenous oxidants to simulate elevated ROS levels.
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http://dx.doi.org/10.1039/d0bm01544jDOI Listing
February 2021

Visualising functional 5-HT receptors containing A and C subunits at or near the cell surface.

Biomed Pharmacother 2020 Dec 12;132:110860. Epub 2020 Oct 12.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC, 3052, Australia; La Trobe Institute for Molecular Science, La Trobe University, PO Box 199, Bendigo, VIC, 3552, Australia. Electronic address:

Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.
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http://dx.doi.org/10.1016/j.biopha.2020.110860DOI Listing
December 2020

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors.

bioRxiv 2020 Oct 6. Epub 2020 Oct 6.

Department for Microbiology and Clinical Immunology, Faculty of Medicine, Tel Aviv University, Israel.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.
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http://dx.doi.org/10.1101/2020.10.06.323634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553166PMC
October 2020

Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A).

Hum Mutat 2020 Jul 11. Epub 2020 Jul 11.

Brain and Mitochondrial Research Group, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.

Defects in the motor domain of kinesin family member 1A (KIF1A), a neuron-specific ATP-dependent anterograde axonal transporter of synaptic cargo, are well-recognized to cause a spectrum of neurological conditions, commonly known as KIF1A-associated neurological disorders (KAND). Here, we report one mutation-negative female with classic Rett syndrome (RTT) harboring a de novo heterozygous novel variant [NP_001230937.1:p.(Asp248Glu)] in the highly conserved motor domain of KIF1A. In addition, three individuals with severe neurodevelopmental disorder along with clinical features overlapping with KAND are also reported carrying de novo heterozygous novel [NP_001230937.1:p.(Cys92Arg) and p.(Pro305Leu)] or previously reported [NP_001230937.1:p.(Thr99Met)] variants in KIF1A. In silico tools predicted these variants to be likely pathogenic, and 3D molecular modeling predicted defective ATP hydrolysis and/or microtubule binding. Using the neurite tip accumulation assay, we demonstrated that all novel KIF1A variants significantly reduced the ability of the motor domain of KIF1A to accumulate along the neurite lengths of differentiated SH-SY5Y cells. In vitro microtubule gliding assays showed significantly reduced velocities for the variant p.(Asp248Glu) and reduced microtubule binding for the p.(Cys92Arg) and p.(Pro305Leu) variants, suggesting a decreased ability of KIF1A to move along microtubules. Thus, this study further expanded the phenotypic characteristics of KAND individuals with pathogenic variants in the KIF1A motor domain to include clinical features commonly seen in RTT individuals.
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http://dx.doi.org/10.1002/humu.24079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908811PMC
July 2020

In the Loop: Extrastriatal Regulation of Spiny Projection Neurons by GPR52.

ACS Chem Neurosci 2020 07 7;11(14):2066-2076. Epub 2020 Jul 7.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria 3010, Australia.

GPR52 is a Gα-coupled orphan receptor identified as a putative target for the treatment of schizophrenia. The unique expression and signaling profile of GPR52 in key areas of dopamine and glutamate dysregulation suggests its activation may resolve both cortical and striatal dysfunction in the disorder. GPR52 mRNA is enriched in the striatum, almost exclusively on dopamine D-expressing medium spiny neurons (MSNs), and to a lesser extent in the cortex, predominantly on D-expressing pyramidal neurons. Synthetic, small molecule GPR52 agonists are effective in preclinical models of psychosis; however, the relative contribution of cortical and striatal GPR52 is unknown. Here we show that the GPR52 agonist, 3-BTBZ, inhibits phencyclidine-induced hyperlocomotor activity to a greater degree than amphetamine-induced motor effects, suggesting a mechanism beyond functional antagonism of striatal dopamine D receptor signaling. Using DARPP-32 phosphorylation and electrophysiological recordings in either striatopallidal or striatonigral MSNs, we were surprised to find no significant effect of 3-BTBZ in striatopallidal MSNs, but GPR52-mediated effects in striatonigral MSNs, where its mRNA is absent. 3-BTBZ increases phosphorylation of T75 on DARPP-32 in striatonigral MSNs, an effect that was dependent on cortical inputs. A similar role for GPR52 in regulating extrastriatal glutamatergic drive onto striatonigral MSNs was also evident in recordings of spontaneous excitatory postsynaptic currents and was shown to be dependent on the metabotropic glutamate (mGlu) receptor subtype 1. Our results demonstrate that GPR52-mediated regulation of striatal function depends heavily on extrastriatal inputs, which may further support its utility as a novel target for the treatment of schizophrenia.
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http://dx.doi.org/10.1021/acschemneuro.0c00197DOI Listing
July 2020

Ptpn6 inhibits caspase-8- and Ripk3/Mlkl-dependent inflammation.

Nat Immunol 2020 01 9;21(1):54-64. Epub 2019 Dec 9.

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.

Ptpn6 is a cytoplasmic phosphatase that functions to prevent autoimmune and interleukin-1 (IL-1) receptor-dependent, caspase-1-independent inflammatory disease. Conditional deletion of Ptpn6 in neutrophils (Ptpn6) is sufficient to initiate IL-1 receptor-dependent cutaneous inflammatory disease, but the source of IL-1 and the mechanisms behind IL-1 release remain unclear. Here, we investigate the mechanisms controlling IL-1α/β release from neutrophils by inhibiting caspase-8-dependent apoptosis and Ripk1-Ripk3-Mlkl-regulated necroptosis. Loss of Ripk1 accelerated disease onset, whereas combined deletion of caspase-8 and either Ripk3 or Mlkl strongly protected Ptpn6 mice. Ptpn6 neutrophils displayed increased p38 mitogen-activated protein kinase-dependent Ripk1-independent IL-1 and tumor necrosis factor production, and were prone to cell death. Together, these data emphasize dual functions for Ptpn6 in the negative regulation of p38 mitogen-activated protein kinase activation to control tumor necrosis factor and IL-1α/β expression, and in maintaining Ripk1 function to prevent caspase-8- and Ripk3-Mlkl-dependent cell death and concomitant IL-1α/β release.
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http://dx.doi.org/10.1038/s41590-019-0550-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923591PMC
January 2020

A pH-responsive nanoparticle targets the neurokinin 1 receptor in endosomes to prevent chronic pain.

Nat Nanotechnol 2019 12 4;14(12):1150-1159. Epub 2019 Nov 4.

Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Nanoparticle-mediated drug delivery is especially useful for targets within endosomes because of the endosomal transport mechanisms of many nanomedicines within cells. Here, we report the design of a pH-responsive, soft polymeric nanoparticle for the targeting of acidified endosomes to precisely inhibit endosomal signalling events leading to chronic pain. In chronic pain, the substance P (SP) neurokinin 1 receptor (NKR) redistributes from the plasma membrane to acidified endosomes, where it signals to maintain pain. Therefore, the NKR in endosomes provides an important target for pain relief. The pH-responsive nanoparticles enter cells by clathrin- and dynamin-dependent endocytosis and accumulate in NKR-containing endosomes. Following intrathecal injection into rodents, the nanoparticles, containing the FDA-approved NKR antagonist aprepitant, inhibit SP-induced activation of spinal neurons and thus prevent pain transmission. Treatment with the nanoparticles leads to complete and persistent relief from nociceptive, inflammatory and neuropathic nociception and offers a much-needed non-opioid treatment option for chronic pain.
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http://dx.doi.org/10.1038/s41565-019-0568-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765343PMC
December 2019

Using a Multiplex Nucleic Acid Hybridization Technique to Determine HCN4 mRNA Expression in the Adult Rodent Brain.

Front Mol Neurosci 2019 28;12:211. Epub 2019 Aug 28.

Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne, VIC, Australia.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels carry a non-selective cationic conductance, I , which is important for modulating neuron excitability. Four genes () encode HCN channels, with each gene having distinct expression and biophysical profiles. Here we use multiplex nucleic acid hybridization to determine HCN4 mRNA expression within the adult mouse brain. We take advantage of this approach to detect HCN4 mRNA simultaneously with either HCN1 or HCN2 mRNA and markers of excitatory (VGlut-positive) and inhibitory (VGat-positive) neurons, which was not previously reported. We have developed a Fiji-based analysis code that enables quantification of mRNA expression within identified cell bodies. The highest HCN4 mRNA expression was found in the habenula (medial and lateral) and the thalamus. HCN4 mRNA was particularly high in the medial habenula with essentially no co-expression of HCN1 or HCN2 mRNA. An absence of I -mediated "sag" in neurons recorded from the medial habenula of knockout mice confirmed that HCN4 channels are the predominant subtype in this region. Analysis in the thalamus revealed HCN4 mRNA in VGlut2-positive excitatory neurons that was always co-expressed with HCN2 mRNA. In contrast, HCN4 mRNA was undetectable in the nucleus reticularis. HCN4 mRNA expression was high in a subset of VGat-positive cells in the globus pallidus external. The majority of these neurons co-expressed HCN2 mRNA while a smaller subset also co-expressed HCN1 mRNA. In the striatum, a small subset of large cells which are likely to be giant cholinergic interneurons co-expressed high levels of HCN4 and HCN2 mRNA. The amygdala, cortex and hippocampus expressed low levels of HCN4 mRNA. This study highlights the heterogeneity of HCN4 mRNA expression in the brain and provides a morphological framework on which to better investigate the functional roles of HCN4 channels.
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http://dx.doi.org/10.3389/fnmol.2019.00211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724756PMC
August 2019

Controlling the size and shape of liposomal ciprofloxacin nanocrystals by varying the lipid bilayer composition and drug to lipid ratio.

J Colloid Interface Sci 2019 Nov 28;555:361-372. Epub 2019 Jul 28.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia; Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia. Electronic address:

Drug nanocrystals precipitated inside liposomes are of increasing interest in liposomal drug delivery. For liposomal nanocrystal formulations, the size and shape of the drug nanocrystals can influence the apparent drug release properties, providing opportunities for developing tailored liposomal drug release systems. Small angle X-ray scattering (SAXS) and quantitative transmission electron microscopy (TEM) can be used to analyse the size distributions of the nanoparticles. In this study, by changing the fluidity of the membrane through the use of different membrane phospholipids with varying cholesterol content, the impact of lipid phase, fluidity and permeability on the size distribution of ciprofloxacin nanocrystals were investigated using standard TEM and SAXS as orthogonal techniques. The results show that the phospholipid phase behaviour has a direct effect on the nanocrystal size distribution, where shorter and thinner nanocrystals were formed in liposomes made from hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids with higher phase transition temperatures than 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with lower transition temperatures. This is mainly due to the phase behaviour of the liposome during nanocrystal formation. The addition of cholesterol that reduces fluidity and permeability of the DOPC liposomes was also shown to restrict the growth of the ciprofloxacin nanocrystals. Moreover, increasing the drug loading of the liposomes made from HSPC and DPPC produced longer and wider nanocrystals. The findings open new opportunities to tailor nanocrystal size distributions, as well as the aspect ratio of the enclosing liposomes with potential to alter drug release and in vivo behaviour.
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http://dx.doi.org/10.1016/j.jcis.2019.07.081DOI Listing
November 2019

Carboxylated Cy5-Labeled Comb Polymers Passively Diffuse the Cell Membrane and Target Mitochondria.

ACS Appl Mater Interfaces 2019 Aug 16;11(34):31302-31310. Epub 2019 Aug 16.

Department of Chemistry , University of Warwick , Coventry CV4 7AL , United Kingdom.

A detailed understanding of the cellular uptake and trafficking of nanomaterials is essential for the design of "smart" intracellular drug delivery vehicles. Typically, cellular interactions can be tailored by endowing materials with specific properties, for example, through the introduction of charges or targeting groups. In this study, water-soluble carboxylated -acylated poly(amino ester)-based comb polymers of different degree of polymerization and side-chain modification were synthesized via a combination of spontaneous zwitterionic copolymerization and redox-initiated reversible addition-fragmentation chain-transfer polymerization and fully characterized by H NMR spectroscopy and size exclusion chromatography. The comb polymers showed no cell toxicity against NIH/3T3 and N27 cell lines nor hemolysis. Detailed cellular association and uptake studies by flow cytometry and confocal laser scanning microscopy (CLSM) revealed that the carboxylated polymers were capable of passively diffusing cell membranes and targeting mitochondria. The interplay of pendant carboxylic acids of the comb polymers and the Cy5-label was identified as major driving force for this behavior, which was demonstrated to be applicable in NIH/3T3 and N27 cell lines. Blocking of the carboxylic acids through modification with 2-methoxyethylamine and poly(2-ethyl-2-oxazoline) or replacement of the dye label with a different dye (e.g., fluorescein) resulted in an alteration of the cellular uptake mechanism toward endocytosis as demonstrated by CLSM. In contrast, partial modification of the carboxylic acid groups allowed to retain the cellular interaction, hence, rendering these comb polymers a highly functional mitochondria targeted carrier platform for future drug delivery applications and imaging purposes.
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http://dx.doi.org/10.1021/acsami.9b09395DOI Listing
August 2019

Activated platelets in the tumor microenvironment for targeting of antibody-drug conjugates to tumors and metastases.

Theranostics 2019 7;9(4):1154-1169. Epub 2019 Feb 7.

Baker Heart and Diabetes Institute, Melbourne, 3004, Australia.

: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFv) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). : We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFv-MMAE was first conjugated with Cyanine7 for imaging. The therapeutic efficacy of the scFv-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. : studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFv-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. : Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.
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http://dx.doi.org/10.7150/thno.29146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401411PMC
January 2020

Direct Comparison of Standard Transmission Electron Microscopy and Cryogenic-TEM in Imaging Nanocrystals Inside Liposomes.

Mol Pharm 2019 04 27;16(4):1775-1781. Epub 2019 Feb 27.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Drug Delivery, Disposition, and Dynamics , Monash Institute of Pharmaceutical Sciences, Monash University , Parkville , VIC 3052 , Australia.

The use of electron microscopy techniques in the understanding of shape and size of nanoparticles are commonly applied to drug nanotechnology, but the type of microscopy and suitability for the particles of interest can have a significant impact on the result. The size and shape of the nanoparticles are crucial in clinical applications; however, direct comparison of the results from standard transmission electron microscopy (TEM) and cryo-TEM have rarely been reported. As a useful case for comparison, liposomal drug nanocrystals are studied here. In this study, the effect of thawing temperature on the size and shape of the ciprofloxacin nanocrystals was determined. A quantitative standard TEM assay was developed to allow for high-throughput particle size analysis. These results were compared to size and shape information obtained using the cryo-TEM method. The results showed broad agreement between the two TEM methods and that ciprofloxacin nanocrystals formed shorter and thinner crystals inside the liposomes at higher thawing temperatures. The results provide confidence in the use of standard TEM to determine the size and shape distribution of solid nanoparticles (in this case, encapsulated inside liposomes) from aqueous media without fear of sample preparation altering the conclusions.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b01308DOI Listing
April 2019

Cell quiescence correlates with enhanced glioblastoma cell invasion and cytotoxic resistance.

Exp Cell Res 2019 01 15;374(2):353-364. Epub 2018 Dec 15.

Department of Surgery (RMH), The University of Melbourne, Parkville VIC 3010, Australia; Department of Microbiology & Immunology, The University of Melbourne, Parkville 3010, Australia. Electronic address:

Glioblastoma (GBM) tumor cells exhibit drug resistance and are highly infiltrative. GBM stem cells (GSCs), which have low proliferative capacity are thought to be one of the sources of resistant cells which result in relapse/recurrence. However, the molecular mechanisms regulating quiescent-specific tumor cell biology are not well understood. Using human GBM cell lines and patient-derived GBM cells, Oregon Green dye retention was used to identify and isolate the slow-cycling, quiescent-like cell subpopulation from the more proliferative cells in culture. Sensitivity of cell subpopulations to temozolomide and radiation, as well as the migration and invasive potential were measured. Differential expression analysis following RNAseq identified genes enriched in the quiescent cell subpopulation. Orthotopic transplantation of cells into mice was used to compare the in vivo malignancy and invasive capacity of the cells. Proliferative quiescence correlated with better TMZ resistance and enhanced cell invasion, in vitro and in vivo. RNAseq expression analysis identified genes involved in the regulation cell invasion/migration and a three-gene signature, TGFBI, IGFBP3, CHI3L1, overexpressed in quiescent cells which correlates with poor GBM patient survival.
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http://dx.doi.org/10.1016/j.yexcr.2018.12.010DOI Listing
January 2019

A Nonionic Polyethylene Oxide (PEO) Surfactant Model: Experimental and Molecular Dynamics Studies of Kolliphor EL.

J Pharm Sci 2019 Jan 28;108(1):193-204. Epub 2018 Nov 28.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052, Australia. Electronic address:

Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6 and 2016H66 force field parameters for polyethoxylate chains. To compare the computational models to experimental measurements, we investigated the phase behavior of KOL using nephelometry, dynamic light scattering, cross-polarized microscopy, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The potential for digestion of KOL was also evaluated using an in vitro digestion experiment. We found that the size and spherical morphology of the KOL colloids at low concentrations was reproduced by the MD models as well as the growing interactions between the aggregates to from rod-like structures at high concentrations. We believe that this model reproduces the phase behavior of KOL relevant to drug absorption and that it can be used in whole formulation simulations to accelerate the formulation development.
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http://dx.doi.org/10.1016/j.xphs.2018.11.028DOI Listing
January 2019

Stress hormone signaling through β-adrenergic receptors regulates macrophage mechanotype and function.

FASEB J 2019 03 3;33(3):3997-4006. Epub 2018 Dec 3.

Department of Integrative Biology and Physiology, University of California, Los Angeles, California, USA.

Critical functions of immune cells require them to rapidly change their shape and generate forces in response to cues from their surrounding environment. However, little is known about how soluble factors that may be present in the microenvironment modulate key aspects of cellular mechanobiology-such as immune cell deformability and force generation-to impact functions such as phagocytosis and migration. Here we show that signaling by soluble stress hormones through β-adrenoceptors (β-AR) reduces the deformability of macrophages; this is dependent on changes in the organization of the actin cytoskeleton and is associated with functional changes in phagocytosis and migration. Pharmacologic interventions reveal that the impact of β-AR signaling on macrophage deformability is dependent on actin-related proteins 2/3, indicating that stress hormone signaling through β-AR shifts actin organization to favor branched structures rather than linear unbranched actin filaments. These findings show that through remodeling of the actin cytoskeleton, β-AR-mediated stress hormone signaling modulates macrophage mechanotype to impact functions that play a critical role in immune response.-Kim, T.-H., Ly, C., Christodoulides, A., Nowell, C. J., Gunning, P. W., Sloan, E. K., Rowat, A. C. Stress hormone signaling through β-adrenergic receptors regulates macrophage mechanotype and function.
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http://dx.doi.org/10.1096/fj.201801429RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404566PMC
March 2019

Changes in duodenal CD163-positive cells in dogs with chronic enteropathy after successful treatment.

Innate Immun 2018 10 17;24(7):400-410. Epub 2018 Sep 17.

1 Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Australia.

Chronic enteropathy (CE) in dogs is characterized retrospectively per treatment response as food-responsive enteropathy (FRE), antibiotic-responsive enteropathy (ARE), and immunosuppressant-responsive enteropathy (IRE) - the latter most resembling inflammatory bowel disease in people. The aim of this study was to characterize duodenal macrophages (Mϕ) in CE using immunohistochemistry; with calprotectin (CAL) as a marker of early differentiated Mϕ and CD163 expression as a marker for resident Mϕ in the duodenum before and after treatment. Prior to treatment, dogs with FRE and IRE had a lower CD163/CAL ratio than control dogs (CTRL) in crypts; this increased significantly and normalized compared with CTRL after treatment. Conversely, the CD163/CAL ratio in dogs with ARE was comparable to that in healthy dogs before and after treatment. In summary, these results suggest that Mϕ play a role in the pathogenesis of CE in FRE and IRE, with a decrease in resident Mϕ and an increase in early differentiated Mϕ, but not in ARE dogs. Mϕ normalize after successful treatment.
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http://dx.doi.org/10.1177/1753425918799865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830873PMC
October 2018