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Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515-518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

Laser-Doppler flow and flow-sensitive magnetic resonance imaging (MRI) were utilized to determine the effects of N-tert-butyl-alpha-phenylnitrone (PBN) on postischemic reperfusion in halothane-anesthetized rats. MRI analysis of postischemic reperfusion in the presence of PBN indicated the role of the free radical trapping agent in attenuating the lesion volume during ischemia in halothane-anesthetized rats. Furthermore, PBN exhibited cytoprotective effects on the vascular endothelium during focal ischemia/reperfusion.

Monte Carlo (MC) based treatment planning solutions for FLASH have typically been confined within proprietary treatment planning systems (TPS), lacking a true open-source implementation. This study aims to establish an open-source treatment planning workflow by integrating the Tool for Particle Simulation (TOPAS) MC code with a Python graphical user interface (GUI) to accurately calculate 3D dose across various electron cut-out apertures and geometries. The treatment head of the FLASH-enabled Linac was modeled using CAD files. Depth dose and beam profiles were acquired using radiographic film with different circular collimators. A beam model was created through inverse Bayesian optimization, minimizing the difference between measured and simulated profiles. The total particle count for each radiation pulse passing through the beam current transformer (BCT) on the mobile electron linear accelerator was estimated from measurements and then the BCT was integrated into the MC simulation, establishing a calibration relationship between MC output, absolute dose and number of histories. The whole workflow was then used for end-to-end testing incorporating an anatomically correct 3D printed mouse phantom. Inverse optimization yielded a beam energy of 8.4 MeV with a spread of 1 MeV and a source spot size of 2 mm with an angular spread of 4 degrees. The simulated and measured depth dose for the 10 cm collimator agreed to within <1% of each other. Simulated beam profiles for various insert sizes exhibited good agreement (<3%) with the measured profiles, although variations were noted in the low dose region for all profiles. A Python GUI was developed enabling the importation of CT scans and beam apertures to calculate 3D dose distributions by invoking the TOPAS MC code for simulation. A recent multi-institutional dosimetric audit uncovered discrepancies of up to 10%, particularly in areas with tissue inhomogeneities. Given the tissue-protective benefits of FLASH radiation are on the order of 10-20%, it is imperative to employ precise planning and delivery tools. Our open-source MC beam model and Python GUI will be invaluable resources for ensuring accuracy in FLASH research, with the potential for seamless adaptation into clinical trials. [ABSTRACT FROM AUTHOR]

Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.Cell Death and Differentiation (2004) 11, 760-770. doi:10.1038/sj.cdd.4401377 Published online 12 March 2004 [ABSTRACT FROM AUTHOR]

To investigate the effects of dopamine (DA) on the release of glutathione (GSH) from astrocytes, we used astroglia-rich primary cultures from the brains of newborn rats. In the absence of DA, GSH accumulated in the medium of these cultures with a constant rate. In contrast, during incubation of the cells with 50 µM DA extracellular GSH was not detectable anymore. This disappearance of extracellular GSH was prevented by superoxide dismutase, indicating that DA does not affect GSH release but rather reacts with the released GSH in a superoxide-dependent reaction. Incubation of astroglial cultures with 0.5 and 1 mM DA established almost constant extracellular concentrations of H[sub 2]O[sub 2] of 5 µM and 15 µM, respectively. Under these conditions astroglial cultures release glutathione disulphide (GSSG). This GSSG export was blocked by catalase and by MK571, an inhibitor of the multidrug resistance protein 1. The effects of DA on the extracellular accumulations of GSH and GSSG were not modulated by inhibitors of DA receptors, DA transport, and monoamine oxidases. The other catecholamines adrenaline and noradrenaline showed similar effects on the accumulation of GSH and GSSG in the medium compared with those obtained for DA. In conclusion, the data presented demonstrate that DA affects astroglial GSH metabolism by two mechanisms: (i) directly by chemical reaction with extracellular GSH, and (ii) indirectly by generation of hydrogen peroxide that leads to the efflux of GSSG from astroglial cells. These observations are discussed in the context of the brain's GSH metabolism in Parkinson's disease. [ABSTRACT FROM AUTHOR]

Inhibitor-of-differentiation 2 (Id2) belongs to a family of transcriptional modulators that are characterized by a helix loop helix region but lack the basic amino acid domain. During development, Id2 antagonizes differentiation mediated by the retinoblastoma protein, probably by scavenging downstream E-box basic helix-loop-helix proteins. Here, using differential display RT-PCR, we identify Id2 as an induced gene during serum and potassium deprivation-induced apoptosis of cerebellar granule neurons. Consistent with a biological role for induced Id2 messenger RNA and protein expression in neuronal cell death, expression of Id2 antisense RNA, or targeted deletion of the Id2 gene in neurons from Id2 knock-out mice, protect from apoptosis. Further, gene transfer- mediated overexpression of Id2 induces neuronal cell death both in high potassium and low potassium conditions. Thus, the present study defines a role for Id2 in the modulation of neuronal apoptosis. [ABSTRACT FROM AUTHOR]

Intrastriatal injection of the reversible succinate dehydrogenase inhibitor malonate results in both chemically induced hypoxia and striatal lesions that are similar to those seen in Huntington's disease and cerebral ischaemia. The mechanisms leading to neuronal death involve secondary excitotoxicity, the release of dopamine from nigrostriatal fibres and the generation of reactive oxygen species (ROS) including nitric oxide (NO) and hydroxyl radicals. Here, we further investigated the contribution and mechanism of dopamine on malonate-induced striatal lesions. Prior lesions of the nigrostriatal pathway with 6-OHDA or the depletion of striatal dopamine stores by pretreatment with reserpine, an inhibitor or the vesicular monoamine transporter type-2 (VMAT2), in combination with α-methyl-p-tyrosine resulted in a significant reduction of malonate-induced striatal lesion volumes. This was paralleled by block or reduction of the malonate-induced generation of ROS, as measured by the conversions of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis. Systemic or intrastriatal application of l-DOPA or dopamine, respectively, reconstituted malonate toxicity and the generation of ROS in 6-OHDA-lesioned rats. Block of the dopamine transporter by GBR12909 did not result in a reduction of malonate-induced dopamine release, but significantly reduced the generation of hydroxyl radicals. The D2 receptor agonist lisuride and the mixed D1 and D2 receptor agonist apomorphine, but not the D1 receptor agonist SKF38393, partially restored malonate toxicity in 6-OHDA-lesioned rats without increasing the generation of ROS. In line with these results sulpiride, an inhibitor of D2 receptors, reduced the malonate-induced lesion volume, whereas SCH23390, an inhbitor of D1 receptors, was ineffective. Our data suggest that malonate-induced dopamine toxicity to energetically impaired neurons is mediated by two independent pathways: (i) dopamine transporter uptake-dependent, dopamine receptor-independent generation of ROS, and (ii) excessive stimulation of D2 receptors. [ABSTRACT FROM AUTHOR]

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H[sub 2]O[sub 2] stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressing brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress. [ABSTRACT FROM AUTHOR]

Longitudinal monitoring of tumor volume is a fundamental component of preclinical biomedical research. We explored the use of inexpensive portable ultrasound (US) probes as a viable, cost-and time-efficient alternative to the standard caliper-based volume measurements in laboratory settings, aiming to compare these methods and establish a more precise measurement technique. 5 BALB/c and 5 C57Bl6/J mice were shaved and injected subcutaneously with 105 colon (CT26) or lung (LLC1) carcinoma cells on the flank. At day 14, tumor length and width (LxW) were measured with either calipers or a portable handheld US probe (frequency: 8-20MHz, maximum depth: 4cm), which provided 2D images across the LxW. Tumors were subsequently excised and imaged with cone-beam computed tomography (CBCT) to obtain a gold-standard volume measurement. The widely-used ellipsoid volume formula was applied to US- and caliper-derived LxW as well as to LxW and the US-derived tumor depth (D) measured from the orthogonal cross-section. In addition to these methods, we developed a novel algorithm designed to estimate tumor volumes from regions of interest (ROIs) defined on two orthogonal US cross sections. The CBCT volumes ranged from 30-170mm3, with an average volume of 82.8 ± 49.6mm3. The average of the caliper-derived volume was 206% ± 76% of the reference volume, the average US-derived volume estimated from the caliper formula was 106% ± 41%, and the average volume from the US-derived LxWxD applied to the traditional ellipsoid formula was 90% ± 34%. The average from the ROI-derived volumes from the 2 orthogonal US views was 94% ± 12%. Our study demonstrates the feasibility of rapid US-based measurement of tumor volumes that offers improved accuracy relative to standard caliper-based approaches. The adoption of a portable handheld US probe for acquiring width, length, and depth measurements enhances the precision of the average tumor volume estimations by ∼100% when applied to the ellipsoid formula. The calculated volumes from the ROIs using two orthogonal US views closely align with this improved performance and exhibit the least variability across tumors, suggesting that this approach is better suited for tumors with irregular shapes. Although image collection with US was initially two times lengthier than caliper measurements, we anticipate that with increased user proficiency, the time required could decrease further. [ABSTRACT FROM AUTHOR]

Introduction Charcot-Marie-Tooth (CMT) disease is a progressively disabling syndrome phenotypically comprising distal muscle weakness and atrophy, foot deformities, sensory loss, and reduced or absent tendon reflexes. In spastic paraplegia (SPG), a hereditary disorder affecting the upper motor neuron only, pareses are spastic, and deep tendon reflexes increased. Mixed forms between both diseases have been previously described ( Liu et al., 2014 ). By multiple gene panel based analysis using next-generation-sequencing (NGS), we herein identified the novel variant c.785T>C; p.Leu262Pro in KIF5A as the putative cause of a mixed CMT and SPG phenotype. Methods The patient’s examination took place in the neuromuscular outpatient clinic of the RWTH Aachen University hospital. After obtaining the patient’s informed consent, the molecular genetic analysis of an NGS-based panel comprising 84 genes in total was performed following the commercialized standard procedures established by CeGaT GmbH. The pathogenicity of each variant was evaluated according to the international ACMG criteria ( Richards et al., 2015 ). Case report At the age of 44 years, the now 48-year-old male patient with a negative family history noticed the first signs of gait unsteadiness. In the course, he developed profound symmetric calf atrophies, high arched feet, claw toes, and steppage gait. Except for the absent ankle jerk reflex, the deep tendon reflexes where surprisingly brisk. Sensory loss turned out comparably mild. Nerve conduction studies showed an axonal motor neuropathy in the legs, but were normal in the arms. The molecular genetic analysis revealed the heterozygous variant c.785T>C; p.Leu262Pro in the KIF5A gene, which has been described in association to SPG10 and/or an axonal CMT subtype following an autosomal dominant inheritance. The variant c.785T>C; p.Leu262Pro affects a highly conserved amino acid position within a functionally relevant domain. A change of secondary protein structure is conceivable as the linear leucine is replaced by proline, a cyclic amino acid. Several other missense mutations are described in the very close vicinity. The variant is predicted to be pathogenic by all available prediction programs. Its allele frequency in the healthy reference population is unknown. Unfortunately, co-segregation analyses could be performed since further family members were not available. For further evaluation, functional tests would be required. Discussion On the current state of knowledge, the novel variant c.785T>C; p.Leu262Pro in KIF5A is evaluated as being a variant of uncertain significance. Due to the well matching phenotype ( Liu et al., 2014 ), however, we consider it to be putatively pathogenic. This example underlines the necessity of precise phenotyping in the context of NGS-based multigene panels. [ABSTRACT FROM AUTHOR]

Commercial devices capable of delivering FLASH radiotherapy are being introduced into radiobiology research. There is a critical necessity for a unified dosimetric protocol to ensure dosimetric consistency across institutes. This work introduces a collaborative, multi-institutional dosimetry protocol designed to validate and standardize dosimetry across two research facilities using FLASH intraoperative systems. Two independent institutes used the same collimator design (4x4 cm2) and same beam parameters to deliver 12 or 14 Gy with FLASH or conventional (CONV) dose rates. For dosimetry, replicates of a realistic anatomy 3D-printed mouse phantom with coronal or sagittal division allowing insertions of radiochromic films, and a set of films (sourced from a singular source) were sent from Institute 1 to Institute 2. The institutes independently calibrated the target doses for FLASH using films from the phantom against the associated measurements from the embedded toroid of the system, and for CONV against the total number of MU. Ultimately, 80 experimental toroid measurements for FLASH (40 per target dose) were acquired in 4 days, with 5 set of films per modality per day of measurement. The films were returned to Institute 1 for analysis and the experimental values for FLASH and CONV were calculated. For 12 Gy target dose, the toroid-derived FLASH doses combined for Institute 1 vs Institute 2 were 11.85 ± 0.11 Gy vs 12.32 ± 0.16 Gy, for 14 Gy were 14.12 ± 0.07 Gy vs 14.09 ± 0.01 Gy. for CONV 11.74 ± 0.23 Gy vs 12.19 ± 0.20 Gy and for 14 Gy were 13.86 ± 0.21 Gy vs 14.47 ± 0.31 Gy. The differences between measured and target doses, as well as between FLASH and CONV doses, were within 3%, and the inter-institutional dose differences were under 5%. Enhancing the protocol could entail incorporating an extra calibration step before the experimental dates to fine tune dose alignment between institutions. The success of the collaborative, multi-institutional dosimetry protocol, using realistic anatomy 3D-printed mouse phantom, is evidenced by the demonstrated consistency in preclinical radiobiological experiments across distinct research facilities. [ABSTRACT FROM AUTHOR]

Abstract: Cap myopathy is a rare congenital myopathy characterized by the presence of caps within muscle fibres and caused by mutations in ACTA1, TPM2 or TPM3. Thus far, only three cases with TPM3-related cap myopathy have been described. Here, we report on the first autosomal dominant family with cap myopathy in three-generations, caused by a novel heterozygous mutation in the alpha-tropomyosin-slow-encoding gene (TPM3; exon 4; c.445C>A; p.Leu149Ile). The three patients experienced first symptoms of muscle weakness in childhood and followed a slowly progressive course. They presented generalized hypotrophy and mild muscle weakness, elongated face, high arched palate, micrognathia, scoliosis and respiratory involvement. Intrafamilial variability of skeletal deformities, respiratory involvement and mild cardiac abnormalities was noted. Muscle MRI revealed a recognizable pattern of fatty muscle infiltration and masseter muscle hypertrophy. Subsarcolemmal caps were present in 6–10% of the fibres and immunoreactive with anti-tropomyosin antibodies. We conclude that the MRI-pattern of muscle involvement and the presence of masseter muscle hypertrophy in cap myopathy may guide molecular genetic diagnosis towards a mutation in TPM3. Regular respiratory examinations are important, even if patients have no anamnestic clues. We compare our findings to all cases of cap myopathy with identified mutations (n =11), thus far reported in the literature. [Copyright &y& Elsevier]

Abstract: The cerebellum and the motor thalamus, connected by cerebellothalamic pathways, are traditionally considered part of the motor-control system. Yet, functional imaging studies and clinical studies including patients with cerebellar disease suggest an involvement of the cerebellum in olfaction. Additionally, there are anecdotal clinical reports of olfactory disturbances elicited by electrical stimulation of the motor thalamus and its neighbouring subthalamic region. Deep brain stimulation (DBS) targeting the cerebellothalamic pathways is an effective treatment for essential tremor (ET), which also offers the possibility to explore the involvement of cerebellothalamic pathways in the sense of smell. This may be important for patient care given the increased use of DBS for the treatment of tremor disorders. Therefore, 21 none-medicated patients with ET treated with DBS (13 bilateral, 8 unilateral) were examined with “Sniffin'' Sticks,” an established and reliable method for olfactory testing. Patients were studied either with DBS switched on and then off or in reversed order. DBS impaired odor threshold and, to a lesser extent, odor discrimination. These effects were sub-clinical as none of the patients reported changes in olfactory function. The findings, however, demonstrate that olfaction can be modulated in a circumscribed area of the posterior (sub-) thalamic region. We propose that the impairment of the odor threshold with DBS is related to effects on an olfacto-motor loop, while disturbed odor discrimination may be related to effects of DBS on short-term memory. [Copyright &y& Elsevier]

LGMD2L is caused by mutations in the anoctamin-5-gene (ANO5). Limb-girdle muscle weakness can also be caused by an immune-mediated necrotizing myopathy (IMNM) with antibodies against 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase (HMGCR), which catalyzes a rate-limiting step in cholesterol biosynthesis and is inhibited by statins. We report on a 44-year-old patient with LGMD2L and HMGCR-associated-IMNM without previous statin exposure. He presented with myalgia, dysphagia and slowly progressive proximal weakness in the legs for four years. CK-levels were permanently increased (5000–7500U/l). HMGCR-antibodies were detected in serum (D-Tek, Mons, Belgium). Electromyography and cardiac exams were normal. Vital capacity was mildly reduced (70%). Muscle biopsy revealed necrotic fibers and basophilic regenerating muscle fibers, without inflammation. Several fibers showed an abnormal MHC-I staining at their membranes. The MAC stain was normal. A whole-body muscle-MRI revealed symmetrical fatty degeneration in hamstrings and medial gastrocnemius muscles, without inflammatory changes. Based on this characteristic MRI pattern, which is unusual for IMNM and has been reported in LGMD2L, direct sequencing of ANO5 was performed and two known pathogenic compound heterozygous mutations were identified. Because of the presence of HMGCR-autoantibodies, oral steroids were started. Six weeks later, the patient suffered no longer from myalgia, the Mingazzini maneuver ameliorated, a swallowing test improved, CK-levels significantly decreased (from 5098 to 1900U/l) and HMGCR-antibodies were no longer detectable. We conclude that it might be useful to screen for additional hereditary causes of necrotizing myopathies in statin-unexposed HMGCR-IMNM patients. Besides the diagnostic challenge, our case indicates the therapeutic dilemma of immunosuppressive treatment regimen in “double trouble necrotizing myopathies” which might be more frequent than previously suspected. [Copyright &y& Elsevier]