Biochemistry and Pharmacology - Research Publications
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ItemVesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation(PUBLIC LIBRARY SCIENCE, 2012-12)Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.
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ItemElevation in Sphingomyelin Synthase Activity Is Associated with Increases in Amyloid-Beta Peptide Generation(PUBLIC LIBRARY SCIENCE, 2013-08-20)A pathological hallmark of Alzheimer's disease (AD) is the presence of amyloid-beta peptide (Aβ) plaques in the brain. Aβ is derived from a sequential proteolysis of the transmenbrane amyloid precursor protein (APP), a process which is dependent on the distribution of lipids present in the plasma membrane. Sphingomyelin is a major membrane lipid, however its role in APP processing is unclear. Here, we assessed the expression of sphingomyelin synthase (SGMS1; the gene responsible for sphingomyelin synthesis) in human brain and found that it was significantly elevated in the hippocampus of AD brains, but not in the cerebellum. Secondly, we assessed the impact of altering SGMS activity on Aβ generation. Inhibition of SGMS activity significantly reduced the level of Aβ in a dose- and time dependent manner. The decrease in Aβ level occurred without changes in APP expression or cell viability. These results when put together indicate that SGMS activity impacts on APP processing to produce Aβ and it could be a contributing factor in Aβ pathology associated with AD.
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ItemISEV position paper: extracellular vesicle RNA analysis and bioinformatics.(Wiley, 2013)Extracellular vesicles (EVs) are the collective term for the various vesicles that are released by cells into the extracellular space. Such vesicles include exosomes and microvesicles, which vary by their size and/or protein and genetic cargo. With the discovery that EVs contain genetic material in the form of RNA (evRNA) has come the increased interest in these vesicles for their potential use as sources of disease biomarkers and potential therapeutic agents. Rapid developments in the availability of deep sequencing technologies have enabled the study of EV-related RNA in detail. In October 2012, the International Society for Extracellular Vesicles (ISEV) held a workshop on "evRNA analysis and bioinformatics." Here, we report the conclusions of one of the roundtable discussions where we discussed evRNA analysis technologies and provide some guidelines to researchers in the field to consider when performing such analysis.
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ItemPresenting evidence-based health information for people with multiple sclerosis: the IN-DEEP project protocol(BMC, 2012-03-16)BACKGROUND: Increasingly, evidence-based health information, in particular evidence from systematic reviews, is being made available to lay audiences, in addition to health professionals. Research efforts have focused on different formats for the lay presentation of health information. However, there is a paucity of data on how patients integrate evidence-based health information with other factors such as their preferences for information and experiences with information-seeking. The aim of this project is to explore how people with multiple sclerosis (MS) integrate health information with their needs, experiences, preferences and values and how these factors can be incorporated into an online resource of evidence-based health information provision for people with MS and their families. METHODS: This project is an Australian-Italian collaboration between researchers, MS societies and people with MS. Using a four-stage mixed methods design, a model will be developed for presenting evidence-based health information on the Internet for people with MS and their families. This evidence-based health information will draw upon systematic reviews of MS interventions from The Cochrane Library. Each stage of the project will build on the last. After conducting focus groups with people with MS and their family members (Stage 1), we will develop a model for summarising and presenting Cochrane MS reviews that is integrated with supporting information to aid understanding and decision making. This will be reviewed and finalised with people with MS, family members, health professionals and MS Society staff (Stage 2), before being uploaded to the Internet and evaluated (Stages 3 and 4). DISCUSSION: This project aims to produce accessible and meaningful evidence-based health information about MS for use in the varied decision making and management situations people encounter in everyday life. It is expected that the findings will be relevant to broader efforts to provide evidence-based health information for patients and the general public. The international collaboration also permits exploration of cultural differences that could inform international practice.
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ItemThe hypoxia imaging agent CuII(atsm) is neuroprotective and improves motor and cognitive functions in multiple animal models of Parkinson's disease(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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ItemMicrowave Synthesis of Prion Protein Fragments up to 111 Amino Acids in Length Generates Biologically Active Peptides(Springer Verlag, 2012)Misfolded conformers of the prion protein are aetiologically implicated in neurodegenerative conditions termed prion diseases (also known as transmissible spongiform encephalopathies). Two constitutively expressed N-terminal peptides corresponding to human residues 23–90 and 23–111 are thought to serve normal physiological roles related to neuronal protection with membrane binding possibly playing a part in their mechanism of action. These peptides, along with several derivatives up to 111 residues in length, have been produced by microwave assisted peptide synthesis. HPLC and MS characterisation showed that the peptides were manufactured in good yields at high purity. Peptides were assayed by fluorescence spectroscopy for synthetic lipid-membrane binding activity and by dichlorodihydrofluorescein diacetate assay for the amelioration of reactive oxygen species production. Results of these assays were similar to those reported for the wild type recombinant PrP, demonstrating that these synthetic peptides are useful for biological and chemical assays of PrP activity. Further, the longest peptide 1–111 was dimerised via a single internal cystine residue with good yield. The high yields and low purification burden of the microwave assisted synthesis method lends itself to the production of difficult to produce peptides for such studies.
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ItemExosomes: vehicles for the transfer of toxic proteins associated with neurodegenerative diseases?(FRONTIERS MEDIA SA, 2012)Exosomes are small membranous vesicles secreted by a number of cell types including neurons and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of endosomes to form multivesicular bodies (MVB). When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell-cell signaling, removal of unwanted proteins, and the transfer of pathogens between cells. One such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Similarly, exosomes are also involved in the processing of the amyloid precursor protein (APP) which is associated with Alzheimer's disease. Exosomes have been shown to contain full-length APP and several distinct proteolytically cleaved products of APP, including Aβ. In addition, these fragments can be modulated using inhibitors of the proteases involved in APP cleavage. These observations provide further evidence for a novel pathway in which PrP and APP fragments are released from cells. Other proteins such as superoxide dismutase I and alpha-synuclein (involved in amyotrophic lateral sclerosis and Parkinson's disease, respectively) are also found associated with exosomes. This review will focus on the role of exosomes in neurodegenerative disorders and discuss the potential of these vesicles for the spread of neurotoxicity, therapeutics, and diagnostics for these diseases.
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ItemThe detection of microRNA associated with Alzheimer's disease in biological fluids using next-generation sequencing technologies.(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.
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ItemDissociation of ERK signalling inhibition from the anti-amyloidogenic action of synthetic ceramide analogues(PORTLAND PRESS LTD, 2012-05)Inhibition of GSL (glycosphingolipid) synthesis reduces Aβ (amyloid β-peptide) production in vitro. Previous studies indicate that GCS (glucosylceramide synthase) inhibitors modulate phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and that the ERK pathway may regulate some aspects of Aβ production. It is not clear whether there is a causative relationship linking GSL synthesis inhibition, ERK phosphorylation and Aβ production. In the present study, we treated CHO cells (Chinese-hamster ovary cells) and SH-SY5Y neuroblastoma cells, that both constitutively express human wild-type APP (amyloid precursor protein) and process this to produce Aβ, with GSL-modulating agents to explore this relationship. We found that three related ceramide analogue GSL inhibitors, based on the PDMP (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) structure, reduced cellular Aβ production and in all cases this was correlated with inhibition of pERK (phosphorylated ERK) formation. Importantly, the L-threo enantiomers of these compounds (that are inferior GSL synthesis inhibitors compared with the D-threo-enantiomers) also reduced ERK phosphorylation to a similar extent without altering Aβ production. Inhibition of ERK activation using either PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] or U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) had no impact on Aβ production, and knockdown of endogenous GCS using small interfering RNA reduced cellular GSL levels without suppressing Aβ production or pERK formation. Our data suggest that the alteration in pERK levels following treatment with these ceramide analogues is not the principal mechanism involved in the inhibition of Aβ generation and that the ERK signalling pathway does not play a crucial role in processing APP through the amyloidogenic pathway.
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ItemPOLYGLUTAMINE AGGREGATION IN HUNTINGTON AND RELATED DISEASES(SPRINGER-VERLAG BERLIN, 2012)Polyglutamine (polyQ)-expansions in different proteins cause nine neurodegenerative diseases. While polyQ aggregation is a key pathological hallmark of these diseases, how aggregation relates to pathogenesis remains contentious. In this chapter, we review what is known about the aggregation process and how cells respond and interact with the polyQ-expanded proteins. We cover detailed biophysical and structural studies to uncover the intrinsic features of polyQ aggregates and concomitant effects in the cellular environment. We also examine the functional consequences ofpolyQ aggregation and how cells may attempt to intervene and guide the aggregation process.