Biochemistry and Pharmacology - Research Publications

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    Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood.
    Cheng, L ; Sharples, RA ; Scicluna, BJ ; Hill, AF (Wiley, 2014)
    INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases. BACKGROUND: Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. METHODS: Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA. CONCLUSION: This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection.
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    Focus on Extracellular Vesicles: Exosomes and Their Role in Protein Trafficking and Biomarker Potential in Alzheimer's and Parkinson's Disease
    Vella, LJ ; Hill, AF ; Cheng, L (MDPI, 2016-02-01)
    Growing evidence indicates that small extracellular vesicles, called exosomes, are prominent mediators of neurodegenerative diseases such as prion, Alzheimer's and Parkinson's disease. Exosomes contain neurodegenerative disease associated proteins such as the prion protein, β-amyloid and α-synuclein. Only demonstrated so far in vivo with prion disease, exosomes are hypothesised to also facilitate the spread of β-amyloid and α-synuclein from their cells of origin to the extracellular environment. In the current review, we will discuss the role of exosomes in Alzheimer's and Parkinson's disease including their possible contribution to disease propagation and pathology and highlight their utility as a diagnostic in neurodegenerative disease.
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    Gene dysregulation is restored in the Parkinson's disease MPTP neurotoxic mice model upon treatment of the therapeutic drug Cu-II(atsm)
    Cheng, L ; Quek, CYJ ; Hung, LW ; Sharples, RA ; Sherratt, NA ; Barnham, KJ ; Hill, AF (NATURE PUBLISHING GROUP, 2016-03-01)
    The administration of MPTP selectively targets the dopaminergic system resulting in Parkinsonism-like symptoms and is commonly used as a mice model of Parkinson's disease. We previously demonstrated that the neuroprotective compound Cu(II)(atsm) rescues nigral cell loss and improves dopamine metabolism in the MPTP model. The mechanism of action of Cu(II)(atsm) needs to be further defined to understand how the compound promotes neuronal survival. Whole genome transcriptomic profiling has become a popular method to examine the relationship between gene expression and function. Substantia nigra samples from MPTP-lesioned mice were evaluated using whole transcriptome sequencing to investigate the genes altered upon Cu(II)(atsm) treatment. We identified 143 genes affected by MPTP lesioning that are associated with biological processes related to brain and cognitive development, dopamine synthesis and perturbed synaptic neurotransmission. Upon Cu(II)(atsm) treatment, the expression of 40 genes involved in promoting dopamine synthesis, calcium signaling and synaptic plasticity were restored which were validated by qRT-PCR. The study provides the first detailed whole transcriptomic analysis of pathways involved in MPTP-induced Parkinsonism. In addition, we identify key therapeutic pathways targeted by a potentially new class of neuroprotective agents which may provide therapeutic benefits for other neurodegenerative disorders.
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    Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells - Evidence of unique microRNA cargos
    Lunavat, TR ; Cheng, L ; Kim, D-K ; Bhadury, J ; Jang, SC ; Lasser, C ; Sharples, RA ; Lopez, MD ; Nilsson, J ; Gho, YS ; Hill, AF ; Lotvall, J (TAYLOR & FRANCIS INC, 2015-08-03)
    Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.
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    Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery
    Quek, C ; Bellingham, SA ; Jung, C-H ; Scicluna, BJ ; Shambrook, MC ; Sharples, RA ; Cheng, L ; Hill, AF (TAYLOR & FRANCIS INC, 2017-01-01)
    Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30-55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.
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    Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors
    Sisquella, X ; Ofir-Birin, Y ; Pimentel, MA ; Cheng, L ; Abou Karam, P ; Sampaio, NG ; Penington, JS ; Connolly, D ; Giladi, T ; Scicluna, BJ ; Sharples, RA ; Waltmann, A ; Avni, D ; Schwartz, E ; Schofield, L ; Porat, Z ; Hansen, DS ; Papenfuss, AT ; Eriksson, EM ; Gerlic, M ; Hill, AF ; Bowie, AG ; Regev-Rudzki, N (NATURE PUBLISHING GROUP, 2017-12-07)
    STING is an innate immune cytosolic adaptor for DNA sensors that engage malaria parasite (Plasmodium falciparum) or other pathogen DNA. As P. falciparum infects red blood cells and not leukocytes, how parasite DNA reaches such host cytosolic DNA sensors in immune cells is unclear. Here we show that malaria parasites inside red blood cells can engage host cytosolic innate immune cell receptors from a distance by secreting extracellular vesicles (EV) containing parasitic small RNA and genomic DNA. Upon internalization of DNA-harboring EVs by human monocytes, P. falciparum DNA is released within the host cell cytosol, leading to STING-dependent DNA sensing. STING subsequently activates the kinase TBK1, which phosphorylates the transcription factor IRF3, causing IRF3 to translocate to the nucleus and induce STING-dependent gene expression. This DNA-sensing pathway may be an important decoy mechanism to promote P. falciparum virulence and thereby may affect future strategies to treat malaria.
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    Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017
    Clayton, A ; Buschmann, D ; Byrd, JB ; Carter, DRF ; Cheng, L ; Compton, C ; Daaboul, G ; Devitt, A ; Manuel Falcon-Perez, J ; Gardiner, C ; Gustafson, D ; Harrison, P ; Helmbrecht, C ; Hendrix, A ; Hill, A ; Hoffman, A ; Jones, JC ; Kalluri, R ; Kang, JY ; Kirchner, B ; Lasser, C ; Lawson, C ; Lenassi, M ; Levin, C ; Llorente, A ; Martens-Uzunova, ES ; Moeller, A ; Musante, L ; Ochiya, T ; Pink, RC ; Tahara, H ; Wauben, MHM ; Webber, JP ; Welsh, JA ; Witwer, KW ; Yin, H ; Nieuwland, R (TAYLOR & FRANCIS LTD, 2018-05-17)
    This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.
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    The hypoxia imaging agent Cu-II(atsm) is neuroprotective and improves motor and cognitive functions in multiple animal models of Parkinson's disease
    Hung, LW ; Villemagne, VL ; Cheng, L ; Sherratt, NA ; Ayton, S ; White, AR ; Crouch, PJ ; Lim, S ; Leong, SL ; Wilkins, S ; George, J ; Roberts, BR ; Pham, CLL ; Liu, X ; Chiu, FCK ; Shackleford, DM ; Powell, AK ; Masters, CL ; Bush, AI ; O'Keefe, G ; Culvenor, JG ; Cappai, R ; Cherny, RA ; Donnelly, PS ; Hill, AF ; Finkelstein, DI ; Barnham, KJ (ROCKEFELLER UNIV PRESS, 2012-04-09)
    Parkinson's disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified Cu(II)(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that Cu(II)(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.
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    The detection of microRNA associated with Alzheimer's disease in biological fluids using next-generation sequencing technologies.
    Cheng, L ; Quek, CYJ ; Sun, X ; Bellingham, SA ; Hill, AF (Frontiers Media SA, 2013)
    Diagnostic tools for neurodegenerative diseases such as Alzheimer's disease (AD) currently involve subjective neuropsychological testing and specialized brain imaging techniques. While definitive diagnosis requires a pathological brain evaluation at autopsy, neurodegenerative changes are believed to begin years before the clinical presentation of cognitive decline. Therefore, there is an essential need for reliable biomarkers to aid in the early detection of disease in order to implement preventative strategies. microRNAs (miRNA) are small non-coding RNA species that are involved in post-transcriptional gene regulation. Expression levels of miRNAs have potential as diagnostic biomarkers as they are known to circulate and tissue specific profiles can be identified in a number of bodily fluids such as plasma, CSF and urine. Recent developments in deep sequencing technology present a viable approach to develop biomarker discovery pipelines in order to profile miRNA signatures in bodily fluids specific to neurodegenerative diseases. Here we review the potential use of miRNA deep sequencing in biomarker identification from biological fluids and its translation into clinical practice.