Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog ...(GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Therapies for Duchenne muscular dystrophy (DMD) must first be tested in animal models to determine proof-of-concept, efficacy, and importantly, safety. The murine and canine models for DMD are ...genetically homologous and most commonly used in pre-clinical testing. Although the mouse is a strong, proof-of-concept model, affected dogs show more analogous clinical and immunological disease progression compared to boys with DMD. As such, evaluating genetic therapies in the canine models may better predict response at the genetic, phenotypic, and immunological levels. We review the use of canine models for DMD and their benefits as it pertains to genetic therapy studies, including gene replacement, exon skipping, and gene editing.
We have examined the effects of intravenous (IV) delivery of rAAVrh74.MHCK7.GALGT2 in the golden retriever muscular dystrophy (GRMD) model of Duchenne Muscular Dystrophy (DMD). After baseline ...testing, GRMD dogs were treated at 3 months of age and reassessed at 6 months. This 3-6 month age range is a period of rapid disease progression, thus offering a relatively short window to establish treatment efficacy. Measures analyzed included muscle AAV transduction, GALGT2 transgene expression, GALGT2-induced glycosylation, muscle pathology, and muscle function. A total of five dogs were treated, 4 at 2x1014vg/kg and one at 6x1014vgkg. The 2x1014vg/kg dose led to transduction of regions of the heart with 1-3 vector genomes (vg) per nucleus, while most skeletal muscles were transduced with 0.25-0.5vg/nucleus. GALGT2-induced glycosylation paralleled levels of myofiber vg transduction, with about 90% of cardiomyocytes having increased glycosylation versus 20-35% of all myofibers across the skeletal muscles tested. Conclusions from phenotypic testing were limited by the small number of dogs. Treated dogs had less pronounced fibrosis and overall lesion severity when compared to control groups, but surprisingly no significant changes in limb muscle function measures. GALGT2-treated skeletal muscle and heart had elevated levels of utrophin protein expression and GALGT2-induced expression of glycosylated α dystroglycan, providing further evidence of a treatment effect. Serum chemistry, hematology, and cardiac function measures were largely unchanged by treatment. Cumulatively, these data show that short-term intravenous treatment of GRMD dogs with rAAVrh74.MHCK7.GALGT2 at high doses can induce muscle glycosylation and utrophin expression and may be safe over a short 3-month interval, but that such treatments had only modest effects on muscle pathology and did not significantly improve muscle strength.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Duchenne muscular dystrophy (DMD) is a lethal, X-chromosome linked muscle-wasting disease affecting about 1 in 3500-6000 boys worldwide. Myofibre necrosis and subsequent loss of muscle mass are due ...to several molecular sequelae, such as inflammation and oxidative stress. We have recently shown increased neutrophils, highly reactive oxidant hypochlorous acid (HOCl) generation by myeloperoxidase (MPO), and associated oxidative stress in muscle from the GRMD dog and mdx mouse models for DMD. These findings have led us to hypothesise that generation of HOCl by myeloperoxidase released from neutrophils has a significant role in dystropathology. Since access to muscle from DMD patients is limited, the aim of this study was to develop methods to study this pathway in urine. Using immunoblotting to measure markers of protein oxidation, we show increased labelling of proteins with antibodies to dinitrophenylhydrazine (DNP, oxidative damage) and DiBrY (halogenation by reactive oxidants from myeloperoxidase) in GRMD and mdx urine. A strong positive correlation was observed between DiBrY labelling in dog urine and muscle. A strong positive correlation was also observed when comparing DNP and DiBrY labelling (in muscle and urine) to markers of dystropathology (plasma creatine kinase) and neutrophil presence (muscle MPO). Our results indicate the presence of neutrophil mediated oxidative stress in both models, and suggest that urine is a suitable bio-fluid for the measurement of such biomarkers. These methods could be employed in future studies into the role of neutrophil mediated oxidative stress in DMD and other inflammatory pathologies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
On western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA A and up to 16% of normal in one dog treated with sgRNA B. There is an error in the ...caption for Fig 6, “Dystrophin expression was minimally restored in HDR-CRISPR-Tx muscle from sgRNA A,” while referring to Miercoles and “Clove.” Scale bar = 50μm. (a) Pre-treatment biopsy sample for Miercoles. (b) HDR-CRISPR injected cranial tibial (CT) Miercoles. (c) HDR-CRISPR injected peroneus longus (PL) Miercoles. (d) HDR-CRISPR injected long digital extensor (LDE) Miercoles. (e) Normal dog muscle. (f) SALINE injected CT Miercoles. (g) SALINE injected PL Miercoles. (h) SALINE injected LDE Miercoles. (i) Pre-treatment biopsy sample for Miercoles. (j) HDR-CRISPR injected LDE Miercoles. (k) Dystrophin intensity quantification for Miercoles and Friendly via One-way ANOVA multiple comparisons test, blue circle indicates CRISPR-Tx limb, black triangle indicates SALINE-Tx limb, gray square indicates pre-treatment biopsied sample; *p<0.05. (l) Normal dog muscle (m) SALINE injected LDE Miercoles. Exon 7 was included in the DMD mRNA of the gene edited muscle. https://doi.org/10.1371/journal.pone.0241430.s002 (TIFF) S8 Fig. Scale bar = 50μm. (a) Pre-treatment biopsy sample for Bubbles (b) HDR-CRISPR injected cranial tibial (CT) Bubbles (c) HDR-CRISPR injected peroneus longus (PL) Bubbles (d) HDR-CRISPR injected long digital extensor (LDE) Bubbles (e) normal dog muscle (f) SALINE injected CT Bubbles (g) SALINE injected PL Bubbles (h) SALINE injected LDE Bubbles. (i) Pre-treatment biopsy sample for Bubbles (j) HDR-CRISPR injected CT Bubbles (k) dystrophin intensity quantification for Bubbles and Clove via One-way ANOVA multiple comparisons test, blue circle indicates CRISPR-Tx limb, black triangle indicates Saline-Tx limb, gray square indicates pre-treatment biopsied sample; *p<0.05 (l) normal dog muscle (m) SALINE injected CT Bubbles.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Considering the recent exponential growth in the amount of information processed in Big Data, the high energy consumed by data processing engines in datacenters has become a major issue, underlining ...the need for efficient resource allocation for more energy-efficient computing. We previously proposed the Best Trade-off Point (BToP) method, which provides a general approach and techniques based on an algorithm with mathematical formulas to find the best trade-off point on an elbow curve of performance vs. resources for efficient resource provisioning in Hadoop MapReduce. The BToP method is expected to work for any application or system which relies on a trade-off elbow curve, non-inverted or inverted, for making good decisions. In this paper, we apply the BToP method to the emerging cluster computing framework, Apache Spark, and show that its performance and energy consumption are better than Spark with its built-in dynamic resource allocation enabled. Our Spark-Bench tests confirm the effectiveness of using the BToP method with Spark to determine the optimal number of executors for any workload in production environments where job profiling for behavioral replication will lead to the most efficient resource provisioning.
The paper presents a novel approach and algorithm with mathematical formula for obtaining the exact optimal number of task resources for any workload running on Hadoop MapReduce. In the era of Big ...Data, energy efficiency has become an important issue for the ubiquitous Hadoop MapReduce framework. However, the question of what is the optimal number of tasks required for a job to get the most efficient performance from MapReduce still has no definite answer. Our algorithm for optimal resource provisioning allows users to identify the best trade-off point between performance and energy efficiency on the runtime elbow curve fitted from sampled executions on the target cluster for subsequent behavioral replication. Our verification and comparison show that the currently well-known rules of thumb for calculating the required number of reduce tasks are inaccurate and could lead to significant waste of computing resources and energy with no further improvement in execution time.
•Overprovisioning could lead to significant waste of computing resources and energy.•Performance gain decreases quickly beyond the best trade-off point on elbow curve.•Our algorithm for optimal resource provisioning is better than any rules of thumbs.•Use dynamic job profiling with table of signatures to match optimal task resources.•Efficient task provisioning saves energy and resources for jobs in multi-tenancy.
Transformation and cancer growth are regulated by the coordinate actions of oncogenes and tumor suppressors. Here, we show that the novel E3 ubiquitin ligase HACE1 is frequently downregulated in ...human tumors and maps to a region of chromosome 6q21 implicated in multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late-onset cancer. A second hit from either environmental triggers or genetic heterozygosity of another tumor suppressor, p53, markedly increased tumor incidence in a Hace1-deficient background. Re-expression of HACE1 in human tumor cells directly abrogates in vitro and in vivo tumor growth, whereas downregulation of HACE1 via siRNA allows non-tumorigenic human cells to form tumors in vivo. Mechanistically, the tumor-suppressor function of HACE1 is dependent on its E3 ligase activity and HACE1 controls adhesion-dependent growth and cell cycle progression during cell stress through degradation of cyclin D1. Thus, HACE1 is a candidate chromosome 6q21 tumor-suppressor gene involved in multiple cancers.
The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin
, a rod-like protein
that protects striated myocytes from contraction-induced injury
. Dystrophin-related protein (or ...utrophin) retains most of the structural and protein binding elements of dystrophin
. Importantly, normal thymic expression in DMD patients
should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.
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