Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD

Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos.

This study investigated the effects of quantity and type of diet fed to superovulated donor heifers on molecular and metabolic indices of embryonic development. These effects included the relative abundances of mRNAs for the alpha 1 subunit of Na/K-ATPase and the antioxidant enzyme Cu/Zn-SOD, as well as pyruvate utilization in bovine morulae and blastocysts developed in vivo. Heifers were fed a daily ration of either grass silage and a citrus-beet pulp-based concentrate or grass silage and a barley-based concentrate for 116 days, both at 3 kg per day or ad libitum. In embryos derived from heifers fed the pulp-based diets, the relative abundances of the transcripts were not affected by either day of collection or quantity of diet. In embryos derived from heifers fed the barley-based diets, the relative abundances of the Na/K-ATPase transcripts were also not changed by these main effects, while the relative abundances of the Cu/Zn-SOD transcripts were affected by day of collection and by the quantity of diet. Pyruvate metabolism was affected by day of collection, and was significantly increased in day 8 embryos compared with day 7 and day 6 embryos. Diet quantity did not affect pyruvate utilization, whereas diet type did increase pyruvate metabolism in the barley group when compared with the pulp group. The results of this study show for the first time that molecular and metabolic variations may exist in embryos derived in vivo and developed in donor heifers on nutritional regimens differing in type and quantity. Differences in embryos collected on different developmental days may be attributed to varying cell numbers. Alterations in the relative abundances of the Cu/Zn-SOD transcripts and pyruvate metabolism caused by the quantity of diet fed to the donor animal were likely to have been due to alterations in metabolic end products that accumulate in reproductive tract fluids, whereas differences in embryonic metabolism caused by type of diet are related to the composition of the diet. These findings characterize embryos produced in vivo at the molecular level, indicating that the molecular markers used in the present study can differentiate between populations of embryos produced under different nutritional regimens and determine conditions conductive to the production of good quality embryos

Muscle RING-finger 2 and 3 maintain striated-muscle structure and function

Background: The Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo. Methods: MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses. Results: DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts. Conclusions: The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo

A Simple and Practical Approach to Unit Testing: The JML and JUnit Way

Writing unit test code is labor-intensive, hence it is often not done as an integral part of programming. However, unit testing is a practical approach to increasing the correctness and quality of software; for example, the Extreme Programming approach relies on frequent unit testing. In this paper we present a new approach that makes writing unit tests easier. It uses a formal specification language\u27s runtime assertion checker to decide whether methods are working correctly, thus automating the writing of unit test oracles. These oracles can be easily combined with hand-written test data. Instead of writing testing code, the programmer writes formal specifications (e.g., pre- and postconditions). This makes the programmer\u27s task easier, because specifications are more concise and abstract than the equivalent test code, and hence more readable and maintainable. Furthermore, by using specifications in testing, specification errors are quickly discovered, so the specifications are more likely to provide useful documentation and inputs to other tools. We have implemented this idea using the Java Modeling Language (JML) and the JUnit testing framework, but the approach could be easily implemented with other combinations of formal specification languages and unit test tools

A Simple and Practical Approach to Unit Testing: The JML and JUnit Way

Writing unit test code is labor-intensive, hence it is often not done as an integral part of programming. However, unit testing is a practical approach to increasing the correctness and quality of software; for example, the Extreme Programming approach relies on frequent unit testing. In this paper we present a new approach that makes writing unit tests easier. It uses a formal specification language\u27s runtime assertion checker to decide whether methods are working correctly, thus automating the writing of unit test oracles. These oracles can be easily combined with hand-written test data. Instead of writing testing code, the programmer writes formal specifications (e.g., pre- and postconditions). This makes the programmer\u27s task easier, because specifications are more concise and abstract than the equivalent test code, and hence more readable and maintainable. Furthermore, by using specifications in testing, specification errors are quickly discovered, so the specifications are more likely to provide useful documentation and inputs to other tools. We have implemented this idea using the Java Modeling Language (JML) and the JUnit testing framework, but the approach could be easily implemented with other combinations of formal specification languages and unit test tools

Mammalian Frataxin: An Essential Function for Cellular Viability through an Interaction with a Preformed ISCU/NFS1/ISD11 Iron-Sulfur Assembly Complex

Frataxin, the mitochondrial protein deficient in Friedreich ataxia, a rare autosomal recessive neurodegenerative disorder, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis.. Fe-S cluster biosynthesis

In Vivo Analysis of MEF2 Transcription Factors in Synapse Regulation and Neuronal Survival

MEF2 (A–D) transcription factors govern development, differentiation and maintenance of various cell types including neurons. The role of MEF2 isoforms in the brain has been studied using in vitro manipulations with only MEF2C examined in vivo. In order to understand specific as well as redundant roles of the MEF2 isoforms, we generated brain-specific deletion of MEF2A and found that Mef2aKO mice show normal behavior in a range of paradigms including learning and memory. We next generated Mef2a and Mef2d brain-specific double KO (Mef2a/dDKO) mice and observed deficits in motor coordination and enhanced hippocampal short-term synaptic plasticity, however there were no alterations in learning and memory, Schaffer collateral pathway long-term potentiation, or the number of dendritic spines. Since previous work has established a critical role for MEF2C in hippocampal plasticity, we generated a Mef2a, Mef2c and Mef2d brain-specific triple KO (Mef2a/c/dTKO). Mef2a/c/d TKO mice have early postnatal lethality with increased neuronal apoptosis, indicative of a redundant role for the MEF2 factors in neuronal survival. We examined synaptic plasticity in the intact neurons in the Mef2a/c/d TKO mice and found significant impairments in short-term synaptic plasticity suggesting that MEF2C is the major isoform involved in hippocampal synaptic function. Collectively, these data highlight the key in vivo role of MEF2C isoform in the brain and suggest that MEF2A and MEF2D have only subtle roles in regulating hippocampal synaptic function

Identification of gene co-regulatory modules and associated cis-elements involved in degenerative heart disease

<p>Abstract</p> <p>Background</p> <p>Cardiomyopathies, degenerative diseases of cardiac muscle, are among the leading causes of death in the developed world. Microarray studies of cardiomyopathies have identified up to several hundred genes that significantly alter their expression patterns as the disease progresses. However, the regulatory mechanisms driving these changes, in particular the networks of transcription factors involved, remain poorly understood. Our goals are (A) to identify modules of co-regulated genes that undergo similar changes in expression in various types of cardiomyopathies, and (B) to reveal the specific pattern of transcription factor binding sites, <it>cis</it>-elements, in the proximal promoter region of genes comprising such modules.</p> <p>Methods</p> <p>We analyzed 149 microarray samples from human hypertrophic and dilated cardiomyopathies of various etiologies. Hierarchical clustering and Gene Ontology annotations were applied to identify modules enriched in genes with highly correlated expression and a similar physiological function. To discover motifs that may underly changes in expression, we used the promoter regions for genes in three of the most interesting modules as input to motif discovery algorithms. The resulting motifs were used to construct a probabilistic model predictive of changes in expression across different cardiomyopathies.</p> <p>Results</p> <p>We found that three modules with the highest degree of functional enrichment contain genes involved in myocardial contraction (n = 9), energy generation (n = 20), or protein translation (n = 20). Using motif discovery tools revealed that genes in the contractile module were found to contain a TATA-box followed by a CACC-box, and are depleted in other GC-rich motifs; whereas genes in the translation module contain a pyrimidine-rich initiator, Elk-1, SP-1, and a novel motif with a GCGC core. Using a naïve Bayes classifier revealed that patterns of motifs are statistically predictive of expression patterns, with odds ratios of 2.7 (contractile), 1.9 (energy generation), and 5.5 (protein translation).</p> <p>Conclusion</p> <p>We identified patterns comprised of putative <it>cis</it>-regulatory motifs enriched in the upstream promoter sequence of genes that undergo similar changes in expression secondary to cardiomyopathies of various etiologies. Our analysis is a first step towards understanding transcription factor networks that are active in regulating gene expression during degenerative heart disease.</p