http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11770 Background: MicroRNA (miRNA) are capable of regulating multitudes of target genes and are essential factors in mediating healthy neurodevelopment. We hypothesize that abnormal miRNA levels contribute to the complex global changes in gene expression that underlie the pathophysiology of schizophrenia. Methods: With a commercial bead array platform, we investigated miRNA expression in 74 samples of postmortem dorsolateral prefrontal cortex (Brodmann Area 46) (n = 37 matched pairs schizophrenia/schizoaffective disorder and control subjects). A subset of differentially expressed miRNA and genes in the miRNA biogenesis pathway was also analyzed with quantitative reverse transcription-polymerase chain reaction. Gene targets of miRNAs demonstrating significantly altered expression were predicted, and pathways analysis was performed. Results: After correction for multiple testing, microarray analysis identified differential expression of 28 miRNA in the schizophrenia group. Significantly, 89% of these molecules were elevated in accordance with earlier work in other brain regions that showed a broad increase in miRNA expression in schizophrenia. These observations were supported by quantitative reverse transcription-polymerase chain reaction, for miR-328, miR-17-5p, miR-134, miR-652, miR-382, and miR-107 and were consistent with a schizophrenia-associated increase in miRNA processing through elevated Dicer expression. Target and pathways analysis provided insight into the potential cellular effects, with particular enrichment of miRNA targets in axon guidance and long-term potentiation. Conclusions: These results suggest that schizophrenia is associated with altered miRNA biogenesis and expression, which might have important implications in the complex pathophysiology of the disorder. 2012-10-19T02:10:10.920Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11111 The advent of gene silencing siRNA technology has created opportunities to develop therapeutics based on targeting the genomics of the disease state. Amongst the first applications of siRNA technology, antiviral applications have been quickly and extensively exploited allowing emergence of a range of antiviral therapeutic strategies. Patent activity has encompassed a range of the components required to utilize this technology ranging from the identification of susceptible genomic targets through to the development of vector systems to express the siRNA endogenously or the synthesis of stable RNA oligonucleotides for in vivo therapeutics. Indeed the primary focus of research effort in this area has been to overcome the challenge common to all of gene therapeutics - delivery of the oligonucleotide - to the diseased tissues and organs, sites of infection and/or sites of drug action. Here, we survey the development of siRNA therapeutics both in terms of the range of virus species targeted and the strategic approaches employed. Our study illustrates features commonly observed in the field of nucleic acid drug development. While in vitro studies provide a broad range of molecules and molecular targets for potential therapeutics, the field is severely limited in terms of safe, effective means to deliver the potential siRNA therapeutics in vivo, to the intracellular site of action. 2012-07-19T23:10:06.849Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11060 Rapid increase in drug-resistant influenza virus isolates, and pandemic threat posed by highly pathogenic avian influenza A and swine flu viruses provide clear and compelling reasons for fast tracking development of novel antiviral drugs. Nucleic acid-based drugs represent a promising class of novel antiviral agents that can be designed to target various seasonal, pandemic and avian influenza viruses. Nucleic acids can be designed to elicit broad-spectrum antiviral responses in the host, by suppressing viral gene expression, or by inducing cleavage or degradation of viral RNA. Immunomodulating nucleic acids, such as double stranded RNA and CpG oligonucleotides, can be potent anti-influenza agents that work by eliciting protective innate and adaptive immunity in the host. By activating the toll-like receptor signaling pathways, these drugs can activate the host's antiviral and inflammatory defenses to combat influenza viruses. Antisense oligonucleotides, small interfering RNAs (siRNA), and nanoRNAs represent sequence specific gene-silencing approaches that could be deployed to suppress or inhibit viral protein gene expression. Lastly, catalytic nucleic acids such as DNAzymes and/or ribozymes can suppress viral replication by repeatedly cleaving viral mRNAs and template RNAs. In summary, nucleic acid-based antiviral agents are versatile, diverse and could complement existing antiviral drugs in combating influenza. 2012-07-09T06:17:43.639Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7723 RNA-mediated gene silencing, in the form of RNA interference (RNAi) or microRNAs (miRNAs) has provided novel tools for gene discovery and validation in mammalian cells. Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells. The library is based in a retroviral vector and uses convergent promoters to produce unique small complementary RNAs. Using this library, we identify a range of small RNA-encoding gene inserts that overcome resistance to 5-fluorouracil (5-FU)- or tumour necrosis factor alpha (TNF-α)- induced cell death in colorectal cancer cells. We demonstrate the utility of this technology platform by identifying a key RNA effector, in the form of a siRNA, which overcomes cell death induced by the chemotherapeutic 5-FU. The technology described has the potential to identify both functional RNA modulators capable of altering physiological systems and the cellular target genes altered by these modulators. 2012-01-30T05:23:48.984Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7836 Background: Custom solid phase oligonucleotide synthesis is an important foundation supporting nearly every aspect of current genomics. In spite of the demand for oligonucleotide primers, their synthesis remains relatively expensive, time consuming and in many circumstances a wasteful process. In this methodology, described as polymerase mediated oligonucleotide synthesis (PMOS), a DNA polymerase is used to increase the hybridization affinity of one oligonucleotide by using another as a template for DNA synthesis. This self-assembly process provides an opportunity to instantly generate a very large number of useful gene-specific primers from a small library of simple precursors. PMOS can be used to generate primers directly in the end-users laboratory within the context of any DNA polymerase chemistry such as in PCR or sequencing reactions. Results: To demonstrate the utility of PMOS, a universal 768-member oligonucleotide library (UniSeq) was designed, fabricated and its performance optimized and evaluated in a range of PCR and DNA sequencing reactions. This methodology used to derive specific 11-mers, performed well in each of these activities and produced the desired amplification or sequencing analysis with results comparable to primers made by time consuming and expensive custom synthesis. Conclusion: On the basis of these experiments, we believe this novel system would be broadly applicable and could in many circumstances replace the need for conventional oligonucleotide synthesis. 2012-01-30T05:13:13.499Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7080 Cisplatin analogues with an attached DNA binding moiety have a higher affinity for DNA, but often suffer from poor aqueous solubility. In this study we examined the DNA sequence specificity of more soluble cisplatin analogues containing the maltolato leaving group in both purified DNA and in intact human cells. In both environments the DNA sequence specificity of these analogues was very similar to cisplatin. However, in purified DNA a higher concentration of the two maltolato-containing analogues was needed to achieve a similar level of DNA damage as cisplatin. This difference in reactivity was not observed in intact cells as the two maltolato-containing complexes were capable of producing a similar level of damage as cisplatin at comparable concentrations. This was consistent with the IC50 values obtained for both cisplatin and the maltolato compounds which were also similar. This study indicated that maltolato can be utilised as the leaving group to increase the aqueous solubility of cisplatin analogues without reducing their biological activity. 2012-01-30T05:02:44.897Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9567 It is well established that Multiple Sclerosis (MS) is an immune mediated disease. Little is known about what drives the differential control of the immune system in MS patients compared to unaffected individuals. MicroRNAs (miRNAs) are small non-coding nucleic acids that are involved in the control of gene expression. Their potential role in T cell activation and neurodegenerative disease has recently been recognised and they are therefore excellent candidates for further studies in MS. We investigated the transcriptome of currently known miRNAs using miRNA microarray analysis in peripheral blood samples of 59 treatment naive MS patients and 37 controls. Of these 59, 18 had a primary progressive, 17 a secondary progressive and 24 a relapsing remitting disease course. In all MS subtypes miR-17 and miR-20a were significantly under-expressed in MS, confirmed by RT-PCR. We demonstrate that these miRNAs modulate T cell activation genes in a knock-in and knock-down T cell model. The same T cell activation genes are also up-regulated in MS whole blood mRNA, suggesting these miRNAs or their analogues may provide useful targets for new therapeutic approaches. 2011-12-05T23:10:03.206Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:9530 Three platinum complexes in which substituted (7-OMe, 9-NH₂; 7-F, 9-NH₂; and 7-H, 9-NH(CH₂)₂OH) 9-aminoacridine-4-carboxamides were tethered to a platinum(II)diamine moiety were synthesised and characterised at the chemical and biological level. These variants showed a decrease in cytotoxicity, as measured by IC₅₀ values in HeLa cells, when compared with the parent 7-H, 9-NH₂ compound. The 7-F and 9-NH(CH₂)₂OH substituents gave rise to a small decrease in cytotoxicity, and the 7-OMe substituent resulted in a larger decrease in cytotoxicity. Their binding to purified pUC19 plasmid DNA was investigated and it was found that the addition of 7-F, 9-NH(CH₂)₂OH and especially the 7-OMe substituents, resulted in reduced DNA binding. This correlated well with the IC₅₀ cytotoxicity values. However, the DNA sequence selectivity was unaffected by the addition of these moieties. 2011-11-29T04:30:05.222Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:7882 Whole-genome microRNA and gene expression analyses were used to monitor changes during retinoic acid induced differentiation of neuroblasts in vitro. Interestingly, the entire miR-17 family was over-represented among the down-regulated miRNA. The implications of these changes are considerable, as target gene prediction suggests that the miR-17 family is involved in the regulation of the mitogen-activated protein kinase (MAPK) signaling pathway, synaptic plasticity and other markers of neuronal differentiation. Significantly, many of the target responses predicted by changes in miRNA expression were supported by the observed changes in gene expression. As expected, markers of neuronal differentiation such as anti-apoptotic protein B-cell lymphoma 2 (BCL2), myocyte enhancer factor-2D (MEF2D) and zipper protein kinase (MAP3K12; aka ZPK/MUK/DLK) were each up-regulated in response to differentiation. The expression of these genes was also reduced in response to miR-17 and miR-20a transfection, and more specifically they were also shown to contain functional miRNA recognition elements for members of the miR-17 family by reporter gene assay. This suggests that the miR-17 family have an integral role in fine-tuning the pathways involved in the regulation of neuronal differentiation. 2011-06-15T23:40:06.769Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5407 Analysis of global microRNA (miRNA) expression in postmortem cortical grey matter from the superior temporal gyrus, revealed significant up-regulation of miR-181b expression in schizophrenia. This finding was supported by quantitative real-time RT-PCR analysis of miRNA expression in a cohort of 21 matched pairs of schizophrenia and non-psychiatric controls. The implications of this finding are substantial, as this miRNA is predicted to regulate many target genes with potential significance to the development of schizophrenia. They include the calcium sensor gene visinin-like 1 (VSNL1) and the ionotropic AMPA glutamate receptor subunit (GRIA2), which were found to be down-regulated in the same cortical tissue from the schizophrenia group. Both of these genes were also suppressed in miR-181b transfected cells and shown to contain functional miR-181b miRNA recognition elements by reporter gene assay. This study suggests altered miRNA levels could be a significant factor in the dysregulation of cortical gene expression in schizophrenia. 2010-04-27T04:48:25.037Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5389 Small double stranded RNA molecules known as small interfering RNA (siRNA), initially identified for their role in the guide sequence in the effector complex of the RNA interference pathway, now represent a promising new class of therapeutic agent with potentially important clinical applications for the prevention and treatment of viral disease. siRNA with its ability to harness the cells own gene-silencing apparatus in a highly adaptable and sequence specific manner has demonstrated unprecedented anti-gene activity. This review highlights the key chemical and biological parameters of this technology and its application in viral suppression, particularly in the respiratory tract. This treatment site represents both an important opportunity to establish clinical exposure for the technology, and an important challenge to provide an additional layer of protection against highly threatening influenza epidemics and potential pandemics. 2010-04-27T04:37:36.482Z ]]>