Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative condition characteris loss of motor neurones and progressive muscle wasting. There is no diagnostic test fo therefore robust biomarkers would not only be valuable for diagnosis, but also the classification of disease subtypes, monitoring responses to drugs and tracking diseas progression. As regulators of gene expression, microRNAs (miRNAs) are increasingly for diagnostic and prognostic purposes in various disease states with increasing explo in neurodegenerative disorders. We hypothesise that circulating blood based miRNAs serve as biomarkers and use miRNA profiling to determine miRNA signatures from th serum of sporadic (sALS) patients compared to healthy controls and patients with dise that mimic ALS. A number of differentially expressed miRNAs were identified in each patient comparisons. Validation in an additional patient cohort showed that miR-206 a miR-143-3p were increased and miR-374b-5p was decreased compared to controls. A continued change in miRNA expression persisted during disease progression indicatin potential use of these particular miRNAs as longitudinal biomarkers in ALS

DOI 10.1007/s10529-009-0130-2

Validation of extraction methods for total RNA and miRNA from bovine blood prior to quantitative gene expression analyse

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Multistability of signal transduction motifs

Protein domains are the basic units of signalling processes. The mechanisms they are involved in usually follow recurring patterns, such as phosphorylation / dephosphorylation cycles. In this contribution, a set of common motifs was defined and their dynamic models were analysed with respect to number and stability of steady states. In a first step Feinberg's Chemical Reaction Network Theory was used to determine whether a motif can show multistationarity or not. The analysis revealed that, apart from double-step activation motifs including a distributive mechanism, only those motifs involving an autocatalytic reaction can show multistationarity. To further characterise these motifs, a large number of randomly chosen parameter sets leading to bistability was generated, followed by a bifurcation analysis for each parameter set and a statistical evaluation of the results. The statistical results can be used to explore robustness against noise, pointing to the observation that multistationarity {at the single-motif level} may not be a robust property: the range of protein concentrations compatible with multistationarity is fairly narrow. Furthermore, experimental evidence suggests that protein concentrations vary substantially between cells. Considering a motif designed to be a bistable switch, this implies that fluctuation of protein concentrations between cells would prevent a significant proportion of motifs to act as a switch. We consider this work as a first step towards a catalogue of fully characterised signalling modules. copyright: The Institution of Engineering and Technology 2008 [accessed 2013 June 14th

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis

Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2-3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls