Target labelling for the detection and profiling of microRNAs expressed in CNS tissue using microarrays

BACKGROUND: MicroRNAs (miRNA) are a novel class of small, non-coding, gene regulatory RNA molecules that have diverse roles in a variety of eukaryotic biological processes. High-throughput detection and differential expression analysis of these molecules, by microarray technology, may contribute to a greater understanding of the many biological events regulated by these molecules. In this investigation we compared two different methodologies for the preparation of labelled miRNAs from mouse CNS tissue for microarray analysis. Labelled miRNAs were prepared either by a procedure involving linear amplification of miRNAs (labelled-aRNA) or using a direct labelling strategy (labelled-cDNA) and analysed using a custom miRNA microarray platform. Our aim was to develop a rapid, sensitive methodology to profile miRNAs that could be adapted for use on limited amounts of tissue. RESULTS: We demonstrate the detection of an equivalent set of miRNAs from mouse CNS tissues using both amplified and non-amplified labelled miRNAs. Validation of the expression of these miRNAs in the CNS by multiplex real-time PCR confirmed the reliability of our microarray platform. We found that although the amplification step increased the sensitivity of detection of miRNAs, we observed a concomitant decrease in specificity for closely related probes, as well as increased variation introduced by dye bias. CONCLUSION: The data presented in this investigation identifies several important sources of systematic bias that must be considered upon linear amplification of miRNA for microarray analysis in comparison to directly labelled miRNA

Estimating reproductive costs in marine mammal bioenergetic models : a review of current knowledge and data availability

This review was funded by the Office of Naval Research (N000142012392), with support from the Marine Mammal Commission (MMC 19-173).Reproductive costs represent a significant proportion of a mammalian female's energy budget. Estimates of reproductive costs are needed for understanding how alterations to energy budgets, such as those from environmental variation or human activities, impact maternal body condition, vital rates and population dynamics. Such questions are increasingly important for marine mammals, as many populations are faced with rapidly changing and increasingly disturbed environments. Here we review the different energetic costs that marine mammals incur during gestation and lactation and how those costs are typically estimated in bioenergetic models. We compiled data availability on key model parameters for each species across all six marine mammal taxonomic groups (mysticetes, odontocetes, pinnipeds, sirenians, mustelids and ursids). Pinnipeds were the best-represented group regarding data availability, including estimates of milk intake, milk composition, lactation duration, birth mass, body composition at birth and growth. There were still considerable data gaps, particularly for polar species, and good data were only available across all parameters in 45\poor, with some species having little or no data for any parameters, particularly beaked whales. Even for species with moderate data coverage, many parameter estimates were tentative or based on indirect approaches, necessitating reevaluation of these estimates. We discuss mechanisms and factors that affect maternal energy investment or prey requirements during reproduction, such as prey supplementation by offspring, metabolic compensation, environmental conditions and maternal characteristics. Filling the existing data gaps highlighted in this review, particularly for parameters that are influential on bioenergetic model outputs, will help refine reproductive costs estimated from bioenergetic models and better address how and when energy imbalances are likely to affect marine mammal populations.Publisher PDFPeer reviewe

Application of “omics” to Prion Biomarker Discovery

The advent of genomics and proteomics has been a catalyst for the discovery of biomarkers able to discriminate biological processes such as the pathogenesis of complex diseases. Prompt detection of prion diseases is particularly desirable given their transmissibility, which is responsible for a number of human health risks stemming from exogenous sources of prion protein. Diagnosis relies on the ability to detect the biomarker PrPSc, a pathological isoform of the host protein PrPC, which is an essential component of the infectious prion. Immunochemical detection of PrPSc is specific and sensitive enough for antemortem testing of brain tissue, however, this is not the case in accessible biological fluids or for the detection of recently identified novel prions with unique biochemical properties. A complementary approach to the detection of PrPSc itself is to identify alternative, “surrogate” gene or protein biomarkers indicative of disease. Biomarkers are also useful to track the progress of disease, especially important in the assessment of therapies, or to identify individuals “at risk”. In this review we provide perspective on current progress and pitfalls in the use of “omics” technologies to screen body fluids and tissues for biomarker discovery in prion diseases

MicroRNA 146a (miR-146a) Is Over-Expressed during Prion Disease and Modulates the Innate Immune Response and the Microglial Activation State

Increasing evidence supports the involvement of microRNAs (miRNAs) in inflammatory and immune processes in prion neuropathogenesis. MiRNAs are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia. Expression profiling of a variety of central nervous system derived cell-lines revealed that miR-146a is preferentially expressed in cells of microglial lineage. Prominent up-regulation of miR-146a was evident in the microglial cell lines BV-2 following TLR2 or TLR4 activation and also EOC 13.31 via TLR2 that reached a maximum 24–48 hours post-stimulation, concomitant with the return to basal levels of transcription of induced cytokines. Gain- and loss-of-function studies with miR-146a revealed a substantial deregulation of inflammatory response pathways in response to TLR2 stimulation. Significant transcriptional alterations in response to miR-146a perturbation included downstream mediators of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-κB) and the JAK-STAT signaling pathway. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in microglial activation states as well as phagocytic mediators of the oxidative burst such as CYBA and NOS3. Based on our results, we propose a role for miR-146a as a potent modulator of microglial function by regulating the activation state during prion induced neurodegeneration

Activation of p53-regulated pro-apoptotic signaling pathways in PrP-mediated myopathy

<p>Abstract</p> <p>Background</p> <p>We have reported that doxycycline-induced over-expression of wild type prion protein (PrP) in skeletal muscles of Tg(HQK) mice is sufficient to cause a primary myopathy with no signs of peripheral neuropathy. The preferential accumulation of the truncated PrP C1 fragment was closely correlated with these myopathic changes. In this study we use gene expression profiling to explore the temporal program of molecular changes underlying the PrP-mediated myopathy.</p> <p>Results</p> <p>We used DNA microarrays, and confirmatory real-time PCR and Western blot analysis to demonstrate deregulation of a large number of genes in the course of the progressive myopathy in the skeletal muscles of doxycycline-treated Tg(HQK) mice. These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles. Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology.</p> <p>Conclusion</p> <p>The data provides the first <it>in vivo </it>evidence that directly links p53 to a wild type PrP-mediated disease. It is evident that several mechanistic features contribute to the myopathy observed in PrP over-expressing mice and that p53-related apoptotic pathways appear to play a major role.</p

From individual responses to population effects : integrating a decade of multidisciplinary research on blue whales and sonar

Funding: Office of Naval Research (GrantNumber(s): N00014-19-1-2464).As ecosystems transform under climate change and expanding human activities, multidisciplinary integration of empirical research, conceptual frameworks and modelling methods is required to predict, monitor and manage the cascading effects on wildlife populations. For example, exposure to anthropogenic noise can lead to changes in the behaviour and physiology of individual marine mammals, but management is complicated by uncertainties on the long-term effects at a population level. We build on a decade of diverse efforts to demonstrate the strengths of integrating research on multiple stressors for assessing population-level effects. Using the case study of blue whales exposed to military sonar in the eastern north Pacific, we model how behavioural responses and environmental effects induced by climate change affect female survival and reproductive success. Environmental changes were predicted to severely affect vital rates, while the current regime of sonar activities was not. Simulated disturbance had a stronger effect on reproductive success than adult survival, as predicted by life-history theory. We show that information on prey resources is critical for robust predictions, as are data on baseline behavioural patterns, energy budgets, body condition and contextual responses to noise. These results will support effective management of the interactions between sonar operations and blue whales in the study area, while providing pragmatic guidance for future data collection to reduce key uncertainties. Our study provides important lessons for the successful integration of multidisciplinary research to inform the assessment of the effects of noise and other anthropogenic stressors on marine predator populations in the context of a changing environment.Publisher PDFPeer reviewe

Higher Serum Immunoglobulin G3 Levels May Predict the Development of Multiple Sclerosis in Individuals With Clinically Isolated Syndrome

Clinically isolated syndrome (CIS) is a first episode of neurological symptoms that may precede a diagnosis of multiple sclerosis (MS). Therefore, studying individuals with CIS may lead to breakthroughs in understanding the development and pathogenesis of MS. In this study, serum levels of immunoglobulin (Ig)G, IgA, IgM, and IgG1-4 were measured in 20 people with CIS and compared with those in 10 healthy controls (HC) and 8 people with MS. Serum Ig levels in individuals with CIS were compared with (a) the time to their conversion from CIS to MS, (b) serum levels of antibodies to Epstein-Barr virus, (c) frequencies of T regulatory (Treg), T follicular regulatory (Tfr), and B cell subsets, and (d) Treg/Tfr expression of Helios. Serum IgG, IgM, and IgG2 levels were significantly lower in people with CIS than HC, and IgG, IgM, and IgG1 levels were significantly lower in people with CIS than MS. After adjusting for age, sex, and serum 25(OH) vitamin D3 [25(OH)D] levels, CIS was associated with lower serum levels of IgG and IgG2 compared with HC (p = 0.001 and p < 0.001, respectively). People with MS had lower IgG2 levels (p < 0.001) and IgG2 proportions (%IgG; p = 0.007) compared with HC. After adjusting for age, sex, and 25(OH)D, these outcomes remained, in addition to lower serum IgA levels (p = 0.01) and increased IgG3 levels (p = 0.053) in people with MS compared with HC. Furthermore, serum from people with MS had increased proportions of IgG1 and IgG3 (p = 0.03 and p = 0.02, respectively), decreased proportions of IgG2 (p = 0.007), and greater ratios of "upstream" to "downstream" IgG subclasses (p = 0.001) compared with HC. Serum IgG3 proportions (%IgG) from people with CIS correlated with the frequency of plasmablasts in peripheral blood (p = 0.02). Expression of Helios by Treg and Tfr cell subsets from individuals with CIS correlated with levels of serum IgG2 and IgG4. IgG3 levels and proportions of IgG3 (%IgG) in serum at CIS diagnosis were inversely correlated with the time until conversion to MS (p = 0.018 and p < 0.001, respectively), suggesting they may be useful prognostic markers of individuals with CIS who rapidly convert to MS.ST, AJ, and MF-P are recipients of the Multiple Sclerosis Society of Western Australia (MSWA) Postdoctoral Research Fellowship. RL is a recipient of a National Health and Medical Research Council Senior Research Fellowship. This work is funded by a National Health and Medical Research Council Project Grant (ID 1067209)