SUMMARY Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington’s disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of micro-satellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide cross-linking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ~2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture, and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1.
Research shows that approximately 14% of school age children with mild traumatic brain injury (TBI) including sports-related concussions (SRCs) remain symptomatic three months after injury. Advanced imaging studies early after injury have shown evidence of axonal damage, reduced N-acetyl aspartate (NAA) and impaired cerebral blood flow (CBF) in individuals with mild TBI. This study was undertaken to determine whether these techniques can provide valuable information in pediatric SRC patients with persistent post-concussive symptoms. Fifteen pediatric subjects ages 8 to 17 years with persistent post-concussive symptoms were evaluated using perfusion-weighted imaging (PWI), three-dimensional (3D) magnetic resonance spectroscopic imaging, and diffusion tensor imaging (DTI) three to 12 months post-SRC. Data were compared with 15 demographically similar (age, gender, and body mass index) controls. In the bilateral thalami, SRC patients showed reduced CBF (p=0.02 and p=0.02) and relative cerebral blood volume (CBV; p=0.05 and p=0.03), compared with controls. NAA/creatine (Cr) and NAA/choline (Cho) ratios were reduced in the corpus callosum (p=0.003; p=0.05) and parietal white matter (p<0.001; p=0.006) of SRC subjects, compared with controls. Significant differences in DTI metrics differentiated patients with cognitive symptoms, compared with those without cognitive symptoms and controls. Advanced imaging methods detect a spectrum of injury including impaired axonal function, neuronal metabolism and perfusion, suggesting involvement of the neurovascular unit in the presence of persistent symptoms in pediatric SRC patients.
mass stability focal distance focal distance Fig. 1. Our method allows users to optimize designs based on a set of performance metrics. Given a design space and a set of performance evaluation functions, our method automatically extracts the Pareto set-those design points with optimal trade-offs. We represent Pareto points in design and performance space with a set of corresponding manifolds (left). The Pareto-optimal solutions are then embedded to allow interactive exploration of performance trade-offs (right). The mapping from manifolds in performance space back to design space allows designers to explore performance trade-offs interactively while visualizing the corresponding geometry and gaining an understanding of a model's underlying properties.Typical design for manufacturing applications requires simultaneous optimization of conflicting performance objectives: Design variations that improve one performance metric may decrease another performance metric. In these scenarios, there is no unique optimal design but rather a set of designs that are optimal for different trade-offs (called Pareto-optimal). In this work, we propose a novel approach to discover the Pareto front, allowing designers to navigate the landscape of compromises efficiently. Our approach is based on a first-order approximation of the Pareto front, which allows entire neighborhoods rather than individual points on the Pareto front to be captured. In addition to allowing for efficient discovery of the Pareto front and the corresponding mapping to the design space, this approach allows us to represent the entire trade-off manifold as a small collection of patches that comprise a high-quality and piecewise-smooth approximation. We illustrate how this technique can be used for navigating performance trade-offs in computer-aided design (CAD) models.
Patients receiving regular hemodialysis sessions have been known to suffer from fatigue and depression. This experiment was designed to determine the effects of far infrared ray (FIR) stimulation on acupoints of patients suffering from renal failure who are receiving regular hemodialysis. Patients receiving long-term and regular hemodialysis who volunteered for this procedure were randomly selected to undergo either FIR or heat pad (HP) therapy to determine the impact of FIR treatment on these patients. Both the activities of the autonomic nervous system and changes in quality of life were measured before and after treatment to determine the effectiveness of the FIR treatment. Results from this study show that FIR therapy decreases both stress and fatigue levels of these patients. It also stimulates autonomic nervous system (ANS) activity in patients who are diagnosed with end-stage renal disease (ESRD) and are receiving regular hemodialysis (HD). Therefore, benefits of FIR stimulation on these patients are clearly demonstrated in this preliminary study.
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