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Accurate Paediatric Brain Tumour Classification Through Improved Quantitative Analysis of 1H MR Imaging and Spectroscopy.
Multimodality imaging is an emerging research topic in neuro-oncology for its potential of being able to demonstrate tumours in a more comprehensive manner. Diffusion-weighted magnetic resonance imaging (dMRI) and proton magnetic resonance spectroscopy (1H-MRS) allow inferring tissue cellularity and biochemical properties, respectively. Combining dMRI and 1H-MRS may provide more accurate diagnosis for paediatric brain tumours than only one modality. This retrospective study collected 1.5-T clinical 1H-MRS and dMRI from 32 patients to assess paediatric brain tumour classification with combined dMRI and 1H-MRS. Specifically, spectral noise of 1H-MRS was suppressed before calculating metabolite concentrations. Extracted radiomic features were apparent diffusion coefficient (ADC) histogram features through dMRI and metabolite concentrations through 1H-MRS. These features were put together and then ranked according to the multiclass area under the curve (mAUC) and selected for tumour classification through machine learning. Tumours were precisely typed by combining noise-suppressed 1H-MRS and dMRI, and the cross-validated accuracy was improved to be 100% according to naïve Bayes. The finally selected radiomic biomarkers, which showed the highest diagnostic ability, were ADC fifth percentile (mAUC = 0.970), myo-inositol (mAUC = 0.952), combined glutamate and glutamine (mAUC = 0.853), total creatine (mAUC = 0.837) and glycine (mAUC = 0.815). The study indicates combining MR imaging and spectroscopy can provide better diagnostic performance than single-modal imaging.
Immunoassay for pyruvate kinase M1/2 as an Alzheimer's biomarker in CSF.
Alzheimer's disease (AD) is characterized by amyloid-beta plaques and tau tangles in the brain, but these markers alone do not predict disease progression. The intersection of these pathologies with other processes including metabolic changes may contribute to disease progression. Brain glucose metabolism changes are among the earliest detectable events in AD. Pyruvate kinase (PKM) has been implicated as a potential biomarker to track these metabolic changes. We have developed an enzyme-linked immunosorbent assay (ELISA) to assess PKM levels in cerebrospinal fluid (CSF). First, we verified the relationship of CSF PKM levels with cognitive decline, revealing a correlation between elevated CSF PKM levels and accelerated cognitive decline in preclinical AD patients in a tau-dependent manner. We developed the ELISA using two PKM-specific antibodies and validated it through quality control steps, indicating robust quantification of PKM. We showed that ELISA measurements of PKM correlate with mass spectrometry values in matching samples. When tested on an independent cohort, the assay confirmed elevation of PKM in AD. These findings support the use of PKM as a potential biomarker for tracking early metabolic changes in AD, offering a novel tool for investigating metabolic alterations and their intersection with other underlying pathologies in AD progression.
Efficient in vivo targeting of the myocardial scar using Moloney murine leukaemia virus complexed with nanoparticles
AbstractDuring myocardial infarction, native myocardium is replaced by a fibrous scar, impairing cardiac pump function and leading to potentially life‐threatening ventricular tachycardias. Gene therapy‐based targeting of the cardiac scar is essential for short‐ and long‐term treatment of post‐infarct sequelae. However, there are currently no effective methods to target and transduce cardiac (myo)fibroblasts (FB) in vivo. Therefore, Moloney murine leukaemia virus (MMLV) encoding for the fluorescent reporter mCherry complexed with magnetic nanoparticles (MNP) in combination with magnetic steering was tested. This approach strongly increased the transduction rate of FB by four‐fold in vitro. Additionally, injection of MMLV/MNP complexes into the forming scar during exposure of the heart to a magnetic field to increase virus dwell‐time 3 days after left ventricular cryo‐injury, a time when FB proliferation in the infarct peaks, yielded efficient transduction of resident FB. We further assessed the functional impact of overexpressing the gap junction protein connexin 43 (Cx43). MMLV‐mediated Cx43 overexpression (MMLV‐Cx43) in FB increased the formation of functional gap junctions in vitro and substantially lowered post‐cryo‐injury ventricular tachycardia incidence (by 50%), as demonstrated by in vivo electrophysiological testing 2 and 8 weeks after MMLV/MNP injection into the lesion. This anti‐arrhythmic effect was probably the result of a decrease in the heterogeneity of conduction around and across the scar, as observed by optical mapping in isolated hearts overexpressing Cx43. Thus, MMLV/MNP complexes combined with magnetic steering offer an efficient strategy for targeting and transducing FB in vitro and in vivo, allowing modulation of the functional properties of cardiac scars. imageKey points Genetic targeting and efficient transduction of (myo)fibroblasts (FB) in vivo are necessary to modify the properties of cardiac scars for therapeutic benefit. However, this has proven highly inadequate because of a lack of suitable viral vectors. We complexed Moloney murine leukaemia virus (MMLV) with magnetic nanoparticles (MNP) and applied a magnetic field to achieve prominent in vitro transduction of FB. Magnet‐assisted in vivo injections of MMLV/MNP complexes into the developing scar enabled efficient transduction of cardiac tissue‐resident FB. MMLV‐Cx43‐based overexpression of connexin 43 increased the density of functional gap junctions in FB in vitro. Following direct injection of the virus into the developing cardiac scar in a murine cryo‐lesion model, electrophysiological in vivo testing showed that the incidence of ventricular tachycardia was reduced by 50% at 2 and 8 weeks post‐treatment with MMLV. Our approach enables efficient targeting and transduction of FB in the cardiac scar, providing a blueprint for translation.
A missense mutation in zinc finger homeobox‐3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice
AbstractA protein altering variant in the gene encoding zinc finger homeobox‐3 (ZFHX3) has recently been associated with lower BMI in a human genome‐wide association study. We investigated metabolic parameters in mice harboring a missense mutation in Zfhx3 (Zfhx3Sci/+) and looked for altered in situ expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence growth and metabolic effects. One‐year‐old male and female Zfhx3Sci/+ mice weighed less, had shorter body length, lower fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin, and insulin‐like growth factor‐1 (IGF1) concentrations than Zfhx3+/+ littermates. In a second cohort of 9–20‐week‐old males and females, Zfhx3Sci/+ mice ate less than wildtype controls, in proportion to body weight. In a third cohort of female‐only Zfhx3Sci/+ and Zfhx3+/+ mice that underwent metabolic phenotyping from 6 to 14 weeks old, Zfhx3Sci/+ mice weighed less and had lower lean mass and energy expenditure, but fat mass did not differ. We detected increased expression of somatostatin and decreased expression of growth hormone‐releasing hormone and growth hormone‐receptor mRNAs in the arcuate nucleus (ARC). Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal expression of orphan G protein‐coupled receptor Gpr50 was decreased. We demonstrate for the first time an energy balance effect of the Zfhx3Sci mutation, likely by altering expression of key ARC neuropeptides to alter growth, food intake, and energy expenditure.
Decreasing HepG2 Cytotoxicity by Lowering the Lipophilicity of Benzo[d]oxazolephosphinate Ester Utrophin Modulators.
Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC50: 4.17 μM, solubility: 477 μM, mouse hepatocyte T 1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay.
2-Arylbenzo[d]oxazole Phosphinate Esters as Second-Generation Modulators of Utrophin for the Treatment of Duchenne Muscular Dystrophy.
Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds, 30 and 27, led to significantly better in vivo exposure compared to ezutromid and alleviation of the dystrophic phenotype in mdx mice. While 30 was found to have dose-limiting hepatotoxicity, 27 and its enantiomers exhibited limited off-target effects, resulting in a safe profile and highlighting their potential utility as next-generation utrophin modulators suitable for progression toward a future DMD therapy.
A genetic modifier suggests that endurance exercise exacerbates Huntington's disease
Polyglutamine expansions in the huntingtin gene cause Huntington's disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed 'draggen' mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration.
Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety
Objective Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. Methods In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. Results We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. Conclusions OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.
Comprehensive Energy Balance Measurements in Mice.
In mice with altered body composition, establishing whether it is food intake or energy expenditure, or both, that is the major determinant resulting in changed energy balance is important. In order to ascertain where the imbalance is, the acquisition of reproducible data is critical. Therefore, here we provide detailed descriptions of how to determine energy balance in mice. This encompasses protocols for establishing energy intake from home cage measurement of food intake, determining energy lost in feces using bomb calorimetry, and using equations to calculate parameters such as energy intake (EI), digested energy intake (DEI), and metabolisable energy intake (MEI) to determine overall energy balance. We also discuss considerations that should be taken into account when planning these experiments, including diet and sample sizes. © 2016 by John Wiley & Sons, Inc.
Mutations in Mll2, an H3K4 methyltransferase, result in insulin resistance and impaired glucose tolerance in mice.
We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5-14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level.
From mice to humans.
The genomes of many species have now been completely sequenced including human and mouse. Great progress has been made in understanding the complex genetics that underlie diabetes and obesity in human populations. One of the current challenges is the functional identification and characterization of the genes within loci that are being mapped. There are many approaches to this problem and this review outlines the valuable role that the mouse can play. We outline the mouse resources that are available to the research community, including knockouts with conditional potential for every gene, and the efforts of the International Mouse Phenotyping Consortium to attach phenotype information to these genes. We also briefly consider the potential of TALEN technology to tailor-make new mouse models of specific mutations discovered in humans. Finally, we consider the recent progress in characterizing the GWAS genes FTO, TCF7L2, CDKAL1, and SLC30A8 in engineered mouse models.
New mutations at the imprinted Gnas cluster show gene dosage effects of Gsα in postnatal growth and implicate XLαs in bone and fat metabolism but not in suckling.
The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XLαs, XLN1, and ALEX or a double dose of maternally expressed Gsα to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gsα, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gsα and loss of expression of XLαs and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XLαs, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XLαs. This is, to our knowledge, the first report describing a role for XLαs in bone metabolism. We propose that XLαs is involved in the regulation of bone and adipocyte metabolism.
A missense mutation in the non-neural G-protein alpha-subunit isoforms modulates susceptibility to obesity.
OBJECTIVE: The Gnas transcription unit located within an imprinting region encodes several proteins, including the G-protein alpha-subunit, Gsalpha, its isoform XLalphas and their variant truncated neural forms GsalphaN1 and XLN1. Gsalpha and GsalphaN1 are expressed predominantly from the maternally derived allele in some tissues, whereas XLalphas and XLN1 are expressed exclusively from the paternally derived allele. The relative contribution of full-length Gsalpha and XLalphas, and truncated forms GsalphaN1 and XLN1 to phenotype is unknown. The edematous-small point mutation (Oed-Sml) in exon 6 of Gnas lies downstream of GsalphaN1 and XLN1, but affects full-length Gsalpha and XLalphas, allowing us to address the role of full-length Gsalpha and XLalphas. The aim of this study was therefore to determine the metabolic phenotypes of Oed and Sml mice, and to correlate phenotypes with affected transcripts. METHODS: Mice were fed standard or high-fat diets and weighed regularly. Fat mass was determined by DEXA analysis. Indirect calorimetry was used to measure metabolic rate. Glucose was measured in tolerance tests and biochemical parameters in fasted plasma samples. Histological analysis of fat and liver was carried out post mortem. RESULTS: Oed mice are obese on either diet and have a reduced metabolic rate. Sml mice are lean and are resistant to a high-fat diet and have an increased metabolic rate. CONCLUSION: Adult Oed and Sml mice have opposite metabolic phenotypes. On maternal inheritance, the obese Oed phenotype can be attributed to non-functional full-length Gsalpha. In contrast, on paternal inheritance, Sml mice were small and resistant to the development of obesity on a high-fat diet, effects that can be attributed to mutant XLalphas. Thus, the neural isoforms, GsalphaN1 and XLN1, do not appear to play a role in these metabolic phenotypes.