Identification of qPCR reference genes suitable for normalizing gene expression in the mdx mouse model of Duchenne muscular dystrophy

The mdx mouse is the most widely-used animal model of the human disease Duchenne muscular dystrophy, and quantitative PCR analysis of gene expression in the muscles of this animal plays a key role in the study of pathogenesis and disease progression and in evaluation of potential therapeutic interventions. Normalization to appropriate stably-expressed reference genes is essential for accurate quantitative measurement, but determination of such genes is challenging: healthy and dystrophic muscles present very different transcriptional environments, further altering with disease progression and muscle use, raising the possibility that no single gene or combination of genes may be stable under all experimental comparative scenarios. Despite the pedigree of this animal model, this problem remains unaddressed. The aim of this work was therefore to comprehensively assess reference gene suitability in the muscles of healthy and dystrophic mice, identifying reference genes appropriate for specific experimental comparisons, and determining whether an essentially universally-applicable set of genes exists. Using a large sample collection comprising multiple muscles (including the tibialis anterior, diaphragm and heart muscles) taken from healthy and mdx mice at three disease-relevant ages, and a panel of sixteen candidate reference genes (FBXO38, FBXW2, MON2, ZFP91, HTATSF1, GAPDH, ACTB, 18S, CDC40, SDHA, RPL13a, CSNK2A2, AP3D1, PAK1IP1, B2M and HPRT1), we used the geNorm, BestKeeper and Normfinder algorithms to identify genes that were stable under multiple possible comparative scenarios. We reveal that no single gene is stable under all conditions, but a normalization factor derived from multiple genes (RPL13a, CSNK2A2, AP3D1 and the widely-used ACTB) appears suitable for normalizing gene expression in both healthy and dystrophic mouse muscle regardless of muscle type or animal age. We further show that other popular reference genes, including GAPDH, are markedly disease- or muscle-type correlated. This study demonstrates the importance of empirical reference gene identification, and should serve as a valuable resource for investigators wishing to study gene expression in mdx mice

Multilevel Parallelization: Grid Methods for Solving Direct and Inverse Problems

In this paper we present grid methods which we have developed for solving direct and inverse problems, and their realization with different levels of optimization. We have focused on solving systems of hyperbolic equations using finite difference and finite volume numerical methods on multicore architectures. Several levels of parallelism have been applied: geometric decomposition of the calculative domain, workload distribution over threads within OpenMP directives, and vectorization. The run-time efficiency of these methods has been investigated. These developments have been tested using the astrophysics code AstroPhi on a hybrid cluster Polytechnic RSC PetaStream (consisting of Intel Xeon Phi accelerators) and a geophysics (seismic wave) code on an Intel Core i7-3930K multicore processor. We present the results of the calculations and study MPI run-time energy efficiency

Children in military custody

A report written by a delegation of British lawyers on the treatment of Palestinian children under Israeli military law

How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse

Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model

A DFN Approach to Evaluating the Hydrogeological Significance of Lithostatic Unloading in Fractured Strata Around Open-Pit Workings

The majority of open-pit mineral workings are established in hydrogeological environments in which unsaturated drainage or saturated groundwater flow occurs predominantly via discrete fracture networks. Stress relaxation resulting from open-pit mineral extraction can lead to a change in host rock fracture network configuration and fracture hydraulic properties, with the potential to change local hydrogeological characteristics and groundwater flow regimes. Research being undertaken at the University of Leeds is applying a DFN approach to investigate the hydrogeological significance of such effects in relation to methodologies for impact assessment at mineral sites. The paper presents a summary of the research approach and preliminary results. A discrete finite element approach to geomechanical modelling has been undertaken with simulation of DFN evolution in response to lithostatic unloading for a range of pre-existing discontinuity configurations, lithological types and variations in in-situ stress regimes. Preliminary modelling results have provided improved understanding of the vertical and lateral extent of potential DFN response for a range of excavation profiles. Research results will be used to define conditions under which open-pit mineral extraction could lead to hydrogeologically significant change in fracture flow drainage characteristics at a scale relevant to hydrogeological impact assessment for new and existing mineral workings

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD

Equivalent material modelling of fractured rock mass resonance effects

Resonance effects in parallel fractured rock masses are investigated using equivalent material models. The mechanisms of spring resonance and superposition resonance are considered. Both of these resonance mechanisms give rise to resonant frequencies, which represent bands of high transmission. Three different representations of a fractured rock mass are adopted: discrete fractures using special elements in the finite difference mesh; a homogenous equivalent medium representing the weakening to the material caused by the fractures; and a localised equivalent medium applied in the vicinity of fractures. The models are excited by a wide-band source, the response measured and a transfer function generated from the results. Results are compared to the prediction of spring and superposition resonant frequencies calculated using analytical equations. It is found that the discrete and localised equivalent materials give similar results, which match the predictions from the analytical equations for both resonance mechanisms, while the equivalent homogenous medium does not show any resonance effects. Showing that this effect occurs in the appropriate equivalent material model helps future prediction of ground borne vibrations from underground sources, such as railway tunnels, as it gives a greater scope of models which can accurately model the propagation of stress waves through fractured rock masses

Numerical modelling of resonance mechanisms in jointed rocks using transfer functions

Resonance effects in parallel jointed rocks subject to stress waves are investigated using transfer functions, derived from signals generated through numerical modelling. Resonance is important for a range of engineering situations as it identifies the frequency of waves which will be favourably transmitted. Two different numerical methods are used for this study, adopting the finite difference method and the combined discrete element-finite difference method. The numerical models are validated by replicating results from previous studies. The two methods are found to behave similarly and show the same resonance effects; one operating at low frequency and the other operating at relatively high frequency. These resonance effects are interpreted in terms of simple physical systems and analytical equations are derived to predict the resonant frequencies of complex rock masses. Low frequency resonance is shown to be generated by a system synonymous with masses between springs, described as spring resonance, with an equal number of resonant frequencies as the number of blocks. High frequency resonance is generated through superposition of multiple reflected waves developing standing waves within intact blocks, described as superposition resonance. While resonance through superposition has previously been identified, resonance based on masses between springs has not been previously identified in jointed rocks. The findings of this study have implications for future analysis of multiple jointed rock masses, showing that a wave travelling through such materials can induce other modes of propagation of waves, i.e. spring resonance

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib

The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side-effects of ibrutinib. The second generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with Non-Hodgkin Lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide (CRP-XL) and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, while size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited SFKs and that SFKs have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of vWF and FVIII ex vivo by addition of intermediate purity FVIII (haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma vWF and FVIII

Characterisation of the pathogenic effects of the in vivo expression of an ALS-linked mutation in D-amino acid oxidase: Phenotype and loss of spinal cord motor neurons

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disorder characterised by selective loss of motor neurons leading to fatal paralysis. Current therapeutic approaches are limited in their effectiveness. Substantial advances in understanding ALS disease mechanisms has come from the identification of pathogenic mutations in dominantly inherited familial ALS (FALS). We previously reported a coding mutation in D-amino acid oxidase (DAOR199W) associated with FALS. DAO metabolises D-serine, an essential co-agonist at the N-Methyl-D-aspartic acid glutamate receptor subtype (NMDAR). Using primary motor neuron cultures or motor neuron cell lines we demonstrated that expression of DAOR199W, promoted the formation of ubiquitinated protein aggregates, activated autophagy and increased apoptosis. The aim of this study was to characterise the effects of DAOR199W in vivo, using transgenic mice overexpressing DAOR199W. Marked abnormal motor features, e.g. kyphosis, were evident in mice expressing DAOR199W, which were associated with a significant loss (19%) of lumbar spinal cord motor neurons, analysed at 14 months. When separated by gender, this effect was greater in females (26%; p< 0.0132). In addition, we crossed the DAOR199W transgenic mouse line with the SOD1G93A mouse model of ALS to determine whether the effects of SOD1G93A were potentiated in the double transgenic line (DAOR199W/SOD1G93A). Although overall survival was not affected, onset of neurological signs was significantly earlier in female double transgenic animals than their female SOD1G93A littermates (125 days vs 131 days, P = 0.0239). In summary, some significant in vivo effects of DAOR199W on motor neuron function (i.e. kyphosis and loss of motor neurons) were detected which were most marked in females and could contribute to the earlier onset of neurological signs in double transgenic females compared to SOD1G93A littermates, highlighting the importance of recognizing gender effects present in animal models of ALS