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Haverhill, United Kingdom

Johnson G.,Queen Mary, University of London | Ferrini A.,Clinical Pathology Laboratories | Dolan S.K.,National University of Ireland, Maynooth | Nolan T.,Sigma Custom Products | And 3 more authors.
Biomarkers in Medicine | Year: 2014

The incidence of invasive aspergillosis (IA), an opportunistic infection in immunocompromised individuals, is rising, but its early diagnosis remains challenging and treatment options are limited. Hence there is an urgent need to improve existing diagnostic procedures as well as develop novel approaches. The clinical usefulness of galactomannan and β-d-glucan, widely used assays detecting cell-wall antigens of Aspergillus, is unclear and depends on clinicians' awareness of their practical limitations. This leaves room for new methods that utilise genomic, proteomic and metabolomics approaches as well as novel detection procedures, for example point-of-care lateral-flow devices. Each of these strategies has its own limitations and it is likely that a combination of methods will be required to achieve optimal performance for the diagnosis of IA and subsequent appropriate patient management. © 2014 Future Medicine Ltd. Source


Huggett J.F.,LCG Group | Foy C.A.,LCG Group | Benes V.,Genomics Core Facility | Emslie K.,Australian National Measurement Institute | And 11 more authors.
Clinical Chemistry | Year: 2013

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology. © 2013 American Association for Clinical Chemistry. Source


Aiba Y.,University of Texas Southwestern Medical Center | Hu J.,University of Texas Southwestern Medical Center | Liu J.,University of Texas Southwestern Medical Center | Xiang Q.,Sigma Custom Products | And 2 more authors.
Biochemistry | Year: 2013

Unlocked nucleic acid (UNA) is an acyclic analogue of RNA that can be introduced into RNA or DNA oligonucleotides. The increased flexibility conferred by the acyclic structure fundamentally affects the strength of base pairing, creating opportunities for improved applications and new insights into molecular recognition. Here we test how UNA substitutions affect allele-selective inhibition of expression of trinucleotide repeat genes Huntingtin (HTT) and Ataxin-3 (ATX-3). We find that the either the combination of mismatched bases and UNA substitutions or UNA substitutions alone can improve potency and selectivity. Inhibition is potent, and selectivities of >40-fold for inhibiting mutant versus wild-type expression can be achieved. Surprisingly, even though UNA preserves the potential for complete base pairing, the introduction of UNA substitutions at central positions within fully complementary duplexes leads to >19-fold selectivity. Like mismatched bases, the introduction of central UNA bases disrupts the potential for cleavage of substrate by argonaute 2 (AGO2) during gene silencing. UNA-substituted duplexes are as effective as other strategies for allele-selective silencing of trinucleotide repeat disease genes. Modulation of AGO2 activity by the introduction of UNA substitutions demonstrates that backbone flexibility is as important as base pairing for catalysis of fully complementary duplex substrates. UNA can be used to tailor RNA silencing for optimal properties and allele-selective action. © 2013 American Chemical Society. Source


Hu J.,University of Texas Southwestern Medical Center | Liu J.,University of Texas Southwestern Medical Center | Narayanannair K.J.,Alnylam Pharmaceuticals | Lackey J.G.,Alnylam Pharmaceuticals | And 9 more authors.
Biochemistry | Year: 2014

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive neurodegenerative disorder that currently has no curative treatments. DRPLA is caused by an expansion of a CAG trinucleotide repeat region within the protein-encoding sequence of the atrophin-1 (ATN-1) gene. Inhibition of mutant ATN-1 protein expression is one strategy for treating DRPLA, and allele-selective gene silencing agents that block mutant expression over wild-type expression would be lead compounds for therapeutic development. Here we develop an assay for distinguishing mutant from wild-type ATN-1 protein by gel electrophoresis. We use this assay to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) for allele-selective inhibition of ATN-1 protein expression. We observed potent and allele-selective inhibition by RNA duplexes that contain mismatched bases relative to the CAG target and have the potential to form miRNA-like complexes. ss-siRNAs that contained mismatches were as selective as mismatch-containing duplexes. We also report allele-selective inhibition by duplex RNAs containing unlocked nucleic acids or abasic substitutions, although selectivities are not as high. Five compounds that showed >8-fold allele selectivity for mutant ATN-1 were also selective for inhibiting the expression of two other trinucleotide repeat disease genes, ataxin-3 (ATXN-3) and huntingtin (HTT). These data demonstrate that the expanded trinucleotide repeat within ATN-1 mRNA is a potential target for compounds designed to achieve allele-selective inhibition of ATN-1 protein, and one agent may allow the targeting of multiple disease genes. © 2014 American Chemical Society. Source

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