Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids—the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah−/−;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah−/−;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah−/−;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah−/−;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah−/−;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics

Stomatal Opening Involves Polar, Not Radial, Stiffening Of Guard Cells

It has long been accepted that differential radial thickening of guard cells plays an important role in the turgor-driven shape changes required for stomatal pore opening to occur [1-4]. This textbook description derives from an original interpretation of structure rather than measurement of mechanical properties. Here we show, using atomic force microscopy, that although mature guard cells display a radial gradient of stiffness, this is not present in immature guard cells, yet young stomata show a normal opening response. Finite element modeling supports the experimental observation that radial stiffening plays a very limited role in stomatal opening. In addition, our analysis reveals an unexpected stiffening of the polar regions of the stomata complexes, both in Arabidopsis and other plants, suggesting a widespread occurrence. Combined experimental data (analysis of guard cell wall epitopes and treatment of tissue with cell wall digesting enzymes, coupled with bioassay of guard cell function) plus modeling lead us to propose that polar stiffening reflects a mechanical, pectin-based pinning down of the guard cell ends, which restricts increase of stomatal complex length during opening. This is predicted to lead to an improved response sensitivity of stomatal aperture movement with respect to change of turgor pressure. Our results provide new insight into the mechanics of stomatal function, both negating an established view of the importance of radial thickening and providing evidence for a significant role for polar stiffening. Improved stomatal performance via altered cell-wall-mediated mechanics is likely to be of evolutionary and agronomic significance

Hyperpolarised carbon-13 magnetic resonance spectroscopy with [1-13C]ethylpyruvate as a preclinical modality for detecting MS-like lesions and their response to fingolimod treatment in the focal experimental autoimmune encephalomyelitis rat model of multiple sclerosis

Multiple sclerosis (MS) is a demyelinating, immune-mediated disease of the central nervous system characterised by both acute episodes of neurological dysfunction and long-term neurodegeneration. Diagnosis and monitoring rely heavily on imaging, with magnetic resonance imaging (MRI) the present gold-standard modality. Current MRI techniques, however, have poor sensitivity for monitoring the development of pre-demyelinated lesions and in assessing treatment response. In MS lesions, highly active immune cells and dysregulated neuronal metabolism cause a localised metabolic shift towards glycolytic lactate production. Hyperpolarised 13C magnetic resonance spectroscopy (MRS) is able to detect this shift, and here, I have demonstrated the ability of hyperpolarised [1-13C]ethylpyruvate MRS to detect MS-like lesions, and their response to fingolimod treatment, in the focal experimental autoimmune encephalomyelitis rat model of MS. This paves the way for future studies using hyperpolarised [1-13C]ethylpyruvate MR in rodent models of MS, and eventually in patients with MS. In hyperpolarised MR, high levels of signal are generated in 13C-labelled tracer molecules using the process of dynamic nuclear polarisation. This requires the tracer molecule to be mixed with a free radical, which facilitates transfer of electron polarisation to the tracer molecule. To optimise an experimental protocol for this and future hyperpolarised [1-13C]ethylpyruvate MR experiments, I compared build-up times and maximum signal generation using AH111501 and Finland radicals, and demonstrated that the AH111501 radical was superior to the Finland radical in both build-up time and maximum signal generated. Finally, to optimise an anaesthetic protocol for future neuroimaging studies, I compared healthy rats at 100%, 90% and 60% inspired oxygen, and demonstrated reduced cerebral perfusion and dysregulated cerebral metabolism with hyperoxia

Hyperpolarised carbon-13 magnetic resonance spectroscopy with [1-13C]ethylpyruvate as a preclinical modality for detecting MS-like lesions and their response to fingolimod treatment in the focal experimental autoimmune encephalomyelitis rat model of multiple sclerosis

Multiple sclerosis (MS) is a demyelinating, immune-mediated disease of the central nervous system characterised by both acute episodes of neurological dysfunction and long-term neurodegeneration. Diagnosis and monitoring rely heavily on imaging, with magnetic resonance imaging (MRI) the present gold-standard modality. Current MRI techniques, however, have poor sensitivity for monitoring the development of pre-demyelinated lesions and in assessing treatment response. In MS lesions, highly active immune cells and dysregulated neuronal metabolism cause a localised metabolic shift towards glycolytic lactate production. Hyperpolarised 13C magnetic resonance spectroscopy (MRS) is able to detect this shift, and here, I have demonstrated the ability of hyperpolarised [1-13C]ethylpyruvate MRS to detect MS-like lesions, and their response to fingolimod treatment, in the focal experimental autoimmune encephalomyelitis rat model of MS. This paves the way for future studies using hyperpolarised [1-13C]ethylpyruvate MR in rodent models of MS, and eventually in patients with MS. In hyperpolarised MR, high levels of signal are generated in 13C-labelled tracer molecules using the process of dynamic nuclear polarisation. This requires the tracer molecule to be mixed with a free radical, which facilitates transfer of electron polarisation to the tracer molecule. To optimise an experimental protocol for this and future hyperpolarised [1-13C]ethylpyruvate MR experiments, I compared build-up times and maximum signal generation using AH111501 and Finland radicals, and demonstrated that the AH111501 radical was superior to the Finland radical in both build-up time and maximum signal generated. Finally, to optimise an anaesthetic protocol for future neuroimaging studies, I compared healthy rats at 100%, 90% and 60% inspired oxygen, and demonstrated reduced cerebral perfusion and dysregulated cerebral metabolism with hyperoxia.</p

Guard cell shape change and the role of expansins and ARP2/3 in stomatal function

Stomata are microscopic pores that are critical for plant survival by regulating plant gas exchange and water regulation. They do this by moderating the aperture of the pore through the two flanking guard cells, which change shape in response to large changes in internal turgor pressure. The extent to which guard cells change shape is dependent on the anisotropic growth and deformation of the guard cell wall, and it is this shape change that is fundamental to the opening and closing of the stomata. Modellers of guard cell mechanics and function are increasingly realising that guard cell shape is crucial for understanding how guard cells can function under such conditions, yet there has been little detailed characterisation of guard cell shape change. In this thesis, I present a novel method of imaging Arabidopsis guard cells using confocal microscopy and a way of processing these images to create 3D reconstructions of the guard cells. These reconstructions provide novel quantitative data on guard cell volume, surface area and other geometric parameters when the stomata are both open and closed. These data provide a novel insight into the type of shape changes that guard cells undergo to control pore aperture. I then report on a series of genes implicated in the control of guard cell shape, focussing on a subunit of the ARP2/3 complex, and a family of proteins implicated in regulating cell wall extensibility, the expansins. By a combination of mutant analysis and stomatal functional bioassays, as well as thermal imaging and gas exchange analysis, I provide evidence for a role of the ARP2/3 complex and expansins in guard cell function

Sophie Hannah’s Hurting Distance as Crime Trauma Fiction

Rodi-Risberg addresses trauma’s generic border-crossing movement through Sophie Hannah’s socially conscious crime thriller Hurting Distance (2007), a trauma narrative of sexual violence and emotional abuse that can be referred to as crime trauma fiction because it incorporates and blends features of both genres. Rodi-Risberg’s main argument is that crime trauma fiction such as Hannah’s novel represents traumatic experience as politically significant by mobilising affect through its themes of violence as social critique. The chapter concludes that contemporary narratives of crime and trauma such as Hannah’s should be seen as an important locus not only for representing traumatic experience, but also for offering a productive space for acknowledging suffering through the ethical witnessing and politically engaged reading of uncomfortable scenes of violence.peerReviewe