ORCID Profile
0000-0003-1770-7910
Current Organisation
University of Melbourne
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Animal Physiology - Cell | Analytical Chemistry not elsewhere classified | Animal Physiology - Systems | Physiology
Expanding Knowledge in the Chemical Sciences | Nervous System and Disorders | Expanding Knowledge in the Biological Sciences |
Publisher: MDPI AG
Date: 13-01-2022
Abstract: Hempseed (Cannabis sativa L.) cake is a by-product after cold-pressing of oil from the hempseed, which is rich in protein and fiber. This study investigated the effect of hempseed cake incorporation on the physicochemical and antioxidant properties of reconstructed potato chips. Varying levels of hempseed cake (0, 5%, 10%, 15%, and 20%) were added, and the results showed that the addition of hempseed cake at 20% level significantly increased the protein and total dietary fiber content from 2.74 ± 0.62 g/100 g to 9.66 ± 0.28 g/100 g and from 2.76 ± 0.31 g/100 g to 13.57 ± 0.42 g/100 g, respectively. In addition, a 20% reduction in lipid content was observed in the 20% hempseed cake addition group. Furthermore, lightness value (L*) was significantly reduced from 72.23 ± 1.22 to 46.40 ± 1.76, while the hardness was enhanced with a higher level of hempseed cake supplementation in the potato chips s le. Compared with the control (no hempseed cake), the supplement of 20% cake increased the total phenolic content from 0.19 ± 0.01 to 0.26 ± 0.01 mg GAE/g. The ABTS radical scavenging rate was also significantly enhanced with the increased levels of hemp cake. However, the peroxide value and TBARS results showed that the addition of hempseed cake accelerated the lipid oxidation in the s le, possibly due to the highly unsaturated fatty acid residues in the hempseed cake. The results suggested that more research is needed for the incorporation of hempseed cake in potato chips.
Publisher: Cold Spring Harbor Laboratory
Date: 05-01-2018
DOI: 10.1101/243683
Abstract: Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing including the anticipatory treatment of monogenic retinal diseases. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing “kamikaze” CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were designed to target Streptococcus pyogenes Cas9 (SpCas9), after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein, YFP), in the presence of mCherry. Both constructs were packaged into AAV2 vector and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks the expression of SpCas9, the efficacy of YFP gene disruption was quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9 targeting CRISPR/Cas) in transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive “kamikaze” CRISPR/Cas system can be used as a robust tool for refined genome editing in the retina, without compromising on-target efficiency.
Publisher: Mary Ann Liebert Inc
Date: 11-2019
DOI: 10.1089/HUM.2019.021
Abstract: Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of
Publisher: The Company of Biologists
Date: 12-2022
DOI: 10.1242/DMM.049651
Abstract: CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3, with most affected in iduals carrying at least one allele with a 966 bp deletion. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). We differentiated these isogenic iPSCs, and iPSCs from an unrelated healthy control donor, to neurons and identified disease-related changes relating to protein synthesis, trafficking and degradation, and in neuronal activity, which were not apparent in CLN3-corrected or healthy control neurons. CLN3 neurons showed numerous membrane-bound vacuoles containing erse storage material and hyperglycosylation of the lysosomal LAMP1 protein. Proteomic analysis showed increase in lysosomal-related proteins and many ribosomal subunit proteins in CLN3 neurons, accompanied by downregulation of proteins related to axon guidance and endocytosis. CLN3 neurons also had lower electrophysical activity as recorded using microelectrode arrays. These data implicate inter-related pathways in protein homeostasis and neurite arborization as contributing to CLN3 disease, and which could be potential targets for therapy.
Publisher: Wiley
Date: 26-07-2002
DOI: 10.1002/IJC.10554
Abstract: Merkel cells form part of the peripheral neuroendocrine system of the skin and act as mechanoreceptors in touch response. Merkel cell carcinoma (MCC) is a rare, aggressive disease with similarities to small cell lung cancer (SCLC), which is also of neuroendocrine origin. We previously identified a novel DNA binding protein complex specific for MCC suspension cell lines, termed Merkel nuclear factor (MNF) by its binding to the POU-IV family DNA binding consensus sequence. Here we report that MNF contains the POU-IV family member Brn-3c and that Brn-3c is expressed in normal Merkel cells. Additionally, Brn-3c protein reactivity is restricted to a subset of MCC biopsies and is not seen in biopsies revealing adherent, variant cell lines lacking neuroendocrine markers. Recently, proper development of murine Merkel cells was shown to require the proneural basic helix-loop-helix transcription factor, atonal family member, MATH1. We demonstrate a correlation between Brn-3c and HATH1 reactivity in MCC biopsies and cell lines with retention of neuroendocrine phenotype. In SCLC, the related basic helix-loop-helix transcription factor HASH1 is responsible for neuroendocrine phenotype, but HASH1 transcripts were not detected in MCC cell lines. We propose that HATH1 and Brn-3c may form a transcriptional hierarchy responsible for determining neuroendocrine phenotype in Merkel cells and that lack of Brn-3c and/or HATH1 in MCC may indicate a more aggressive disease requiring closer patient follow-up.
Publisher: SAGE Publications
Date: 13-04-2020
Abstract: Because our beliefs regarding our in iduality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the “mini-brain,” the “brain in a dish”. At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell–generated preparation for in vitro modelling (challenges lified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term “dementia.” We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model “dementia in a dish.”
Publisher: MDPI AG
Date: 04-08-2020
DOI: 10.20944/PREPRINTS202008.0091.V1
Abstract: The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer& rsquo s disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch cl . We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.
Publisher: Wiley
Date: 2001
DOI: 10.1002/IJC.1352
Abstract: The majority of small-cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA-D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p14(ARF) antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus.
Publisher: Wiley
Date: 06-07-2015
DOI: 10.1002/BMB.20881
Abstract: Inquiry-based learning (IBL) activities are complementary to the processes of laboratory discovery, as both are focused on producing new findings through research and inquiry. Here, we describe the results of student surveys taken pre- and postpractical to an IBL undergraduate practical on PCR. Our analysis focuses primarily student perceptions of knowledge acquisition and their ability to troubleshoot problems. The survey results demonstrate significant self-reported gains in knowledge related to DNA structure and PCR, and an increase in confidence with "troubleshooting problems during scientific experiments." We conclude that the IBL-based approach that combines PCR primer design with wet laboratory experimentation using student-designed primers, provides students a sense of confidence by imparting workplace and research skills that are integral to erse forms and applications of laboratory practices.
Publisher: Springer Science and Business Media LLC
Date: 14-06-2016
DOI: 10.1007/S10561-016-9563-8
Abstract: The ability to generate human induced pluripotent stem cells (iPSCs) has opened new avenues for human disease modelling and therapy. The aim of our study was to determine research participants' understanding of the information given when donating skin biopsies for the generation of patient-specific iPSCs. A customised 35-item questionnaire based on previous iPSC consent guidelines was sent to participants who had previously donated s les for iPSC research. The questionnaire asked pertinent demographic details, participants' motivation to take part in iPSC research and their attitudes towards related ethical issues. 234 participants were contacted with 141 (60.3 %) complete responses received. The median duration between recruitment and follow-up questioning was 313 days (range 10-573 days). The majority of participants (n = 129, 91.5 %) believed they understood what a stem cell was however, only 22 (16.1 %) correctly answered questions related to basic stem cell properties. We found no statistically significant difference in responses from participants with different levels of education, or those with a health sciences background. The poor understanding amongst participants of iPSC research is unlikely to be unique to our study and may impact future research if not improved. As such, there is a need to develop an easily understood yet comprehensive consent process to ensure ongoing ethical progress of iPSC biobanking.
Publisher: Baishideng Publishing Group Inc.
Date: 2016
Publisher: Springer Science and Business Media LLC
Date: 27-02-2018
Publisher: Elsevier BV
Date: 2018
Publisher: Wiley
Date: 07-04-2011
Publisher: Springer Science and Business Media LLC
Date: 10-08-2016
DOI: 10.1038/SREP30552
Abstract: Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro . In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies.
Publisher: Humana Press
Date: 2006
Publisher: Wiley
Date: 18-08-2004
DOI: 10.1002/IJC.20556
Publisher: MDPI AG
Date: 23-12-2018
DOI: 10.3390/IJMS20010057
Abstract: A contributing factor in the development of ulcerative colitis (UC) and Crohn’s disease (CD) is the disruption of innate and adaptive signaling pathways due to aberrant cytokine production. The cytokine, interleukin (IL)-1β, is highly inflammatory and its production is tightly regulated through transcriptional control and both inflammasome-dependent and inflammasome- independent proteolytic cleavage. In this study, qRT-PCR, immunohistochemistry, immunofluorescence confocal microscopy were used to (1) assess the mRNA expression of NLRP3, IL-1β, CASP1 and ASC in paired biopsies from UC and CD patient, and (2) the colonic localization and spatial relationship of NLRP3 and IL-1β in active and quiescent disease. NLRP3 and IL-1β were found to be upregulated in active UC and CD. During active disease, IL-1β was localized to the infiltrate of lamina propria immune cells, which contrasts with the near-exclusive epithelial cell layer expression during non-inflammatory conditions. In active disease, NLRP3 was consistently expressed within the neutrophils and other immune cells of the lamina propria and absent from the epithelial cell layer. The disparity in spatial localization of IL-1β and NLRP3, observed only in active UC, which is characterized by a neutrophil-dominated lamina propria cell population, implies inflammasome-independent processing of IL-1β. Consistent with other acute inflammatory conditions, these results suggest that blocking both caspase-1 and neutrophil-derived serine proteases may provide an additional therapeutic option for treating active UC.
Publisher: Wiley
Date: 28-12-2013
DOI: 10.1002/CBF.2946
Abstract: Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux 'signatures' during these stress conditions are poorly characterized. We investigated the kinetics of K(+) , Ca(2+) and H(+) ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non-invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K(+) efflux was observed following acute ER stress with peak K(+) efflux being -30·6 and -138·7 nmolm(-2) s(-1) for 10 and 50 µg ml(-1) , respectively (p < 0·01). TM-dependent Ca(2+) uptake was more prolonged with peak values of 0·85 and 2·68 nmol m(-2) s(-1) for 10 and 50 µg ml(-1) TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K(+) efflux and Ca(2+) uptake. While K(+) changes were significantly higher at each time point tested, Ca(2+) uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K(+) efflux and Ca(2+) uptake. Functional assays to investigate the effect of inhibiting K(+) efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K(+) , Ca(2+) and H(+) ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases.
Publisher: Cold Spring Harbor Laboratory
Date: 02-02-2023
DOI: 10.1101/2023.02.01.526555
Abstract: The exact pathogenesis of primary open-angle glaucoma (POAG) is poorly understood. Genome-wide association studies (GWAS) have recently uncovered many loci associated with variation in intraocular pressure (IOP) a crucial risk factor for POAG. Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs), the regulatory cells of IOP. Sixty-two genes at fifty-five loci associated with IOP variation were knocked out in primary TMC lines. All cells underwent high-throughput microscopy imaging after being stained with a five-channel fluorescent cell staining protocol. A convolutional neural network (CNN) was trained to distinguish between gene knockout and normal control cell images. The area under the receiver operator curve (AUC) metric was used to quantify morphological variation in gene knockouts to identify potential pathological perturbations. Cells where RALGPS1 had been perturbed demonstrated the greatest morphological variation from normal TMCs (AUC 0.851, SD 0.030), followed by LTBP2 (AUC 0.846, SD 0.029) and BCAS3 (AUC 0.845, SD 0.020). Of seven multi-gene loci, five had statistically significant differences in AUC (p .05) between genes, allowing for pathological gene prioritisation. The mitochondrial channel most frequently showed the greatest degree of morphological variation (33.9% of cell lines). We demonstrate a robust method for functionally interrogating genome-wide association signals using high-throughput microscopy and AI. Genetic variations inducing marked morphological variation can be readily identified, allowing for the gene-based dissection of loci associated with complex traits.
Publisher: Elsevier BV
Date: 04-2009
DOI: 10.1016/J.BIOCEL.2008.08.037
Abstract: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcriptional regulators. Accumulating evidence suggests that PPARgamma agonists such as the thiazolidinediones (TZDs) may prove to be useful anti-cancer agents exhibiting anti-proliferative and/or pro-apoptotic affects in a range of cancer cell types including melanoma, however, the mechanisms underlying this effect remain unclear. We have demonstrated the anti-proliferative effects of full and partial PPARgamma modulators in human melanoma cell lines. Ablation of PPARgamma expression in the MM96L melanoma cell line by siRNA mediated mechanisms attenuates the anti-proliferative effect of these agents suggesting this effect is directly mediated by PPARgamma. The mechanisms underlying the anti-proliferative effects of PPARgamma in melanoma cells involve the regulation of expression of a number of critical cell cycle genes and beta-catenin. Moreover, our data indicate that PPARgamma modulates Wnt/beta-catenin mediated signalling in melanoma cells in an agonist dependent manner.
Publisher: The Company of Biologists
Date: 28-10-2010
DOI: 10.1242/DMM.005447
Abstract: There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson’s disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson’s disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery.
Publisher: Elsevier BV
Date: 10-2021
Publisher: Cold Spring Harbor Laboratory
Date: 29-04-2022
DOI: 10.1101/2022.04.28.489465
Abstract: CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3 . Most in iduals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient ( CLN3 Δ 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing erse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.
Publisher: Springer Science and Business Media LLC
Date: 02-02-2004
Publisher: Elsevier BV
Date: 12-2021
DOI: 10.1016/J.SCR.2021.102568
Abstract: Multiple sclerosis (MS) is a chronic autoimmune and neurodegenerative disease that results in immune cell infiltration of the central nervous system (CNS) and demyelination in young adults. Substantial progress has been made in developing disease modifying therapies for people with relapsing-remitting MS, but options remain limited for people with primary progressive MS (PPMS). PPMS accounts for ∼15% of MS diagnoses. Herein, we generated a human induced pluripotent stem cell line (hiPSC) from a person with clinically definite PPMS. This disease-specific hiPSC line will be useful for studying PPMS in vitro, allowing the generation of immune and CNS cell types.
Publisher: MDPI AG
Date: 16-01-2023
DOI: 10.3390/IJMS24021766
Abstract: Neurodegenerative diseases present a progressive loss of neuronal structure and function, leading to cell death and irrecoverable brain atrophy. Most have disease-modifying therapies, in part because the mechanisms of neurodegeneration are yet to be defined, preventing the development of targeted therapies. To overcome this, there is a need for tools that enable a quantitative assessment of how cellular mechanisms and erse environmental conditions contribute to disease. One such tool is genetically encodable fluorescent biosensors (GEFBs), engineered constructs encoding proteins with novel functions capable of sensing spatiotemporal changes in specific pathways, enzyme functions, or metabolite levels. GEFB technology therefore presents a plethora of unique sensing capabilities that, when coupled with induced pluripotent stem cells (iPSCs), present a powerful tool for exploring disease mechanisms and identifying novel therapeutics. In this review, we discuss different GEFBs relevant to neurodegenerative disease and how they can be used with iPSCs to illuminate unresolved questions about causes and risks for neurodegenerative disease.
Publisher: Elsevier BV
Date: 08-2005
DOI: 10.1016/J.YEXCR.2005.04.019
Abstract: Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast-melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype.
Publisher: Cold Spring Harbor Laboratory
Date: 10-2023
Publisher: Hindawi Limited
Date: 08-2008
DOI: 10.1002/HUMU.20788
Abstract: The Melanocortin-1 Receptor (MC1R) is a G-protein coupled receptor, which is responsible for production of the darker eumelanin pigment and the tanning response. The MC1R gene has many polymorphisms, some of which have been linked to variation in pigmentation phenotypes within human populations. In particular, the p.D84E, p.R151C, p.R160W and p.D294 H alleles have been strongly associated with red hair, fair skin and increased skin cancer risk. These red hair colour (RHC) variants are relatively well described and are thought to result in altered receptor function, while still retaining varying levels of signaling ability in vitro. The mouse Mc1r null phenotype is yellow fur colour, the p.R151C, p.R160W and p.D294 H alleles were able to partially rescue this phenotype, leading to the question of what the true null phenotype of MC1R would be in humans. Due to the rarity of MC1R null alleles in human populations, they have only been found in the heterozygous state until now. We report here the first case of a homozygous MC1R null in idual, phenotypic analysis indicates that red hair and fair skin is found in the absence of MC1R function.
Publisher: Springer Science and Business Media LLC
Date: 16-03-2021
DOI: 10.1007/S12015-021-10147-5
Abstract: Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimer's disease (AD), with the presence of APOE-ε4 associated with increased risk of developing AD. Here, we reprogrammed human fibroblasts from in iduals with different APOE-ε genotypes into induced pluripotent stem cells (iPSCs), and generated isogenic lines with different APOE profiles. Following characterisation of the newly established iPSC lines, we used an unguided/unpatterning differentiation method to generate six-month-old cerebral organoids from all iPSC lines to assess the suitability of this in vitro system to measure APOE, β amyloid, and Tau phosphorylation levels. We identified variabilities in the organoids' cell composition between cell lines, and between batches of differentiation for each cell line. We observed more homogenous cerebral organoids, and similar levels of APOE, β amyloid, and Tau when using the CRISPR-edited APOE isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-ε4/ε4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-ε status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the cell-line-to-cell-line and batch-to-batch variabilities need to be considered when using cerebral organoids.
Publisher: Cold Spring Harbor Laboratory
Date: 12-2020
DOI: 10.1101/2020.11.30.405654
Abstract: Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimer’s disease, with the presence of APOE-ε4 associated with increased risk of developing Alzheimer’s disease. Here, we reprogrammed human fibroblasts from in iduals with different APOE-ε genotypes into induced pluripotent stem cells, and generated isogenic lines with different APOE profiles. We then differentiated these into cerebral organoids for six months and assessed the suitability of this in vitro system to measure APOE, β amyloid, and Tau phosphorylation levels. We identified intra- and inter-variabilities in the organoids’ cell composition. Using the CRISPR-edited APOE isogenic lines, we observed more homogenous cerebral organoids, and similar levels of APOE, β amyloid, and Tau between the isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-ε4/ε4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-ε status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the batch-to-batch and cell-line-to-cell-line variabilities need to be considered when using cerebral organoids.
Publisher: Wiley
Date: 13-05-2003
DOI: 10.1034/J.1600-0749.2003.00033.X
Abstract: Recent population studies have demonstrated an association with the red-hair and fair-skin phenotype with variant alleles of the melanocortin-1 receptor (MC1R) which result in amino acid substitutions within the coding region leading to an altered receptor activity. In particular, Arg151Cys, Arg160Trp and Asp294His were the most commonly associated variants seen in the south-east Queensland population with at least one of these alleles found in 93% of those with red hair. In order to study the in idual effects of these variants on melanocyte biology and melanocytic pigmentation, we established a series of human melanocyte strains genotyped for the MC1R receptor which included wild-type consensus, variant heterozygotes, compound heterozygotes and homozygotes for Arg151Cys, Arg160Trp, Val60Leu and Val92Met alleles. These strains ranged from darkly pigmented to amelanotic, with all strains of consensus sequence having dark pigmentation. UV sensitivity was found not to be associated with either MC1R genotype or the level of pigmentation with a range of sensitivities seen across all genotypes. Ultrastructural analysis demonstrated that while consensus strains contained stage IV melanosomes in their terminal dendrites, Arg151Cys and Arg160Trp homozygote strains contained only stage II melanosomes. This was despite being able to show expression of tyrosinase and tyrosinase-related protein-1 markers, although at reduced levels and an ability to convert exogenous 3,4-dihydroxyphenyl-alanine (DOPA) to melanin in these strains.
Publisher: MDPI AG
Date: 02-09-2020
DOI: 10.3390/CELLS9092018
Abstract: The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer’s disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch cl . We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.
Publisher: Wiley
Date: 25-02-2011
DOI: 10.1111/J.1600-0854.2011.01172.X
Abstract: Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in melanoma cells quantitatively increased melanosome concentration at the cell periphery. Rab17 knockdown did not inhibit melanosome maturation nor movement, but it caused accumulation of melanin inside cells. Double knockdown of Rab17 and Rab27a indicated that Rab17 acts on melanosomes downstream of Rab27a. Filopodia are known to play a role in melanosome transfer, and in Rab17 knockdown cells filopodia formation was inhibited. Furthermore, we show that stimulation of melanoma cells with α-melanocyte-stimulating hormone induces filopodia formation, supporting a role for filopodia in melanosome release. Cell stimulation also caused redistribution of REs to the periphery, and knockdown of additional RE-associated Rabs 11a and 11b produced a similar accumulation of melanosomes and melanin to that seen after loss of Rab17. Our findings reveal new functions for RE and Rab17 in pigmentation through a distal step in the process of melanosome release via filopodia.
Publisher: Frontiers Media SA
Date: 04-01-2023
DOI: 10.3389/FNAGI.2022.1025473
Abstract: Acetylation is a key post-translational modification (PTM) involved in the regulation of both histone and non-histone proteins. It controls cellular processes such as DNA transcription, RNA modifications, proteostasis, aging, autophagy, regulation of cytoskeletal structures, and metabolism. Acetylation is essential to maintain neuronal plasticity and therefore essential for memory and learning. Homeostasis of acetylation is maintained through the activities of histone acetyltransferases (HAT) and histone deacetylase (HDAC) enzymes, with alterations to these tightly regulated processes reported in several neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS). Both hyperacetylation and hypoacetylation can impair neuronal physiological homeostasis and increase the accumulation of pathophysiological proteins such as tau, α-synuclein, and Huntingtin protein implicated in AD, PD, and HD, respectively. Additionally, dysregulation of acetylation is linked to impaired axonal transport, a key pathological mechanism in ALS. This review article will discuss the physiological roles of protein acetylation and examine the current literature that describes altered protein acetylation in neurodegenerative disorders.
Publisher: Frontiers Media SA
Date: 10-09-2020
Publisher: Cold Spring Harbor Laboratory
Date: 21-06-2020
DOI: 10.1101/2020.06.21.163766
Abstract: The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) - cells that can be differentiated into any cell type of the three germ layers - has provided a foundation for in vitro human disease modelling 1,2 , drug development 1,2 , and population genetics studies 3,4 . In the majority of instances, the expression levels of genes, plays a critical role in contributing to disease risk, or the ability to identify therapeutic targets. However, while the effect of the genetic background of cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of in idual cells. Differences in the effect of genetic variation on the gene expression of different cell-types, would provide significant resolution for in vitro research using preprogramed cells. By bringing together single cell RNA sequencing 15–21 and population genetics, we now have a framework in which to evaluate the cell-types specific effects of genetic variation on gene expression. Here, we performed single cell RNA-sequencing on 64,018 fibroblasts from 79 donors and we mapped expression quantitative trait loci (eQTL) at the level of in idual cell types. We demonstrate that the large majority of eQTL detected in fibroblasts are specific to an in idual sub-type of cells. To address if the allelic effects on gene expression are dynamic across cell reprogramming, we generated scRNA-seq data in 19,967 iPSCs from 31 reprogramed donor lines. We again identify highly cell type specific eQTL in iPSCs, and show that that the eQTL in fibroblasts are almost entirely disappear during reprogramming. This work provides an atlas of how genetic variation influences gene expression across cell subtypes, and provided evidence for patterns of genetic architecture that lead to cell-types specific eQTL effects.
Publisher: Public Library of Science (PLoS)
Date: 12-03-2010
Publisher: Public Library of Science (PLoS)
Date: 07-2011
Publisher: Elsevier BV
Date: 11-2003
Publisher: Cold Spring Harbor Laboratory
Date: 29-08-2023
DOI: 10.1101/2023.08.28.555025
Abstract: The loss of upper and lower motor neurons, and their axons is central to the loss of motor function and death in amyotrophic lateral sclerosis (ALS). Due to the erse range of genetic and environmental factors that contribute to the pathogenesis of ALS, there have been difficulties in developing effective therapies for ALS. One dichotomy emerging in the field is that protection of the neuronal cell soma itself does not prevent axonal vulnerability and degeneration, suggesting the need for targeted therapeutics to prevent axon degeneration. Post-translational modifications of protein acetylation can alter the function, stability and half-life of in idual proteins, and can be enzymatically modified by histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs), which add, or remove acetyl groups, respectively. Maintenance of post-translational microtubule acetylation has been suggested as a potential mechanism to stabilise axons and prevent axonal loss and neurodegeneration in ALS. This study has utilized an orally dosed HDAC6 specific inhibitor, ACY-738, prevent deacetylation and stabilize microtubules in the mSOD1G93A mouse model of ALS. Furthermore, co-treatment with riluzole was performed to determine any effects or drug interactions and potentially enhance preclinical research translation. This study shows ACY-738 treatment increased acetylation of microtubules in the spinal cord of mSOD1G93A mice, reduced lower motor neuron degeneration in the lumbar spinal cord of female mice, ameliorated reduction in peripheral nerve axon puncta size, but did not prevent overt motor function decline. The current study also shows peripheral nerve axon puncta size to be partially restored after treatment with riluzole and highlights the importance of co-treatment to measure the potential effects of therapeutics in ALS.
Publisher: American Society for Microbiology
Date: 05-2014
Abstract: This article assesses the impact of examination mapping as a tool to enhancing assessment and teaching quality in a second-year biochemistry unit for undergraduates. Examination mapping is a process where all questions in a written examination paper are assessed for links to the unit’s intended learning outcomes. We describe how mapping a final written examination helped visualise the impact of the assessment task on intended learning outcomes and skills for that biochemistry unit. The method involved complete analysis of all the final examination questions, and linking each question to task-specific criteria or learning outcomes. This strategy also identified the distribution of marks to key learning outcomes such as knowledge, interpretation and application. Our results indicated that 45% of the questions addressed the knowledge aspect while 37% and 18% were allocated to interpretation and application facets of the intended learning outcomes respectively. In addition, our exam mapping strategy aided in defining the characteristics of examination questions. The examination mapping exercise proved to be a useful process that can enhance a balanced assessment of skills in addition to devising strategies such as criterion-referenced assessment for examinations.
Publisher: Elsevier BV
Date: 10-2021
Publisher: Springer Science and Business Media LLC
Date: 2013
Publisher: American Association for the Advancement of Science (AAAS)
Date: 08-04-2022
Abstract: The human immune system displays substantial variation between in iduals, leading to differences in susceptibility to autoimmune disease. We present single-cell RNA sequencing (scRNA-seq) data from 1,267,758 peripheral blood mononuclear cells from 982 healthy human subjects. For 14 cell types, we identified 26,597 independent cis-expression quantitative trait loci (eQTLs) and 990 trans-eQTLs, with most showing cell type-specific effects on gene expression. We subsequently show how eQTLs have dynamic allelic effects in B cells that are transitioning from naïve to memory states and demonstrate how commonly segregating alleles lead to interin idual variation in immune function. Finally, using a Mendelian randomization approach, we identify the causal route by which 305 risk loci contribute to autoimmune disease at the cellular level. This work brings together genetic epidemiology with scRNA-seq to uncover drivers of interin idual variation in the immune system.
Publisher: Springer Science and Business Media LLC
Date: 05-03-2021
DOI: 10.1186/S13059-021-02293-3
Abstract: The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) has provided a foundation for in vitro human disease modelling, drug development and population genetics studies. Gene expression plays a critical role in complex disease risk and therapeutic response. However, while the genetic background of reprogrammed cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of in idual cells which would provide significant resolution. By integrating single cell RNA-sequencing (scRNA-seq) and population genetics, we apply a framework in which to evaluate cell type-specific effects of genetic variation on gene expression. Here, we perform scRNA-seq on 64,018 fibroblasts from 79 donors and map expression quantitative trait loci (eQTLs) at the level of in idual cell types. We demonstrate that the majority of eQTLs detected in fibroblasts are specific to an in idual cell subtype. To address if the allelic effects on gene expression are maintained following cell reprogramming, we generate scRNA-seq data in 19,967 iPSCs from 31 reprogramed donor lines. We again identify highly cell type-specific eQTLs in iPSCs and show that the eQTLs in fibroblasts almost entirely disappear during reprogramming. This work provides an atlas of how genetic variation influences gene expression across cell subtypes and provides evidence for patterns of genetic architecture that lead to cell type-specific eQTL effects.
Publisher: Wiley
Date: 11-2016
DOI: 10.1111/CEO.12837
Start Date: 2020
End Date: 2022
Funder: Australian Research Council
View Funded ActivityStart Date: 2017
End Date: 2017
Funder: University of Tasmania Foundation Inc
View Funded ActivityStart Date: 2017
End Date: 2017
Funder: University of Tasmania Foundation Inc
View Funded ActivityStart Date: 2019
End Date: 2022
Funder: FightMND
View Funded ActivityStart Date: 11-2020
End Date: 12-2024
Amount: $545,563.00
Funder: Australian Research Council
View Funded Activity