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Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development. [ABSTRACT FROM AUTHOR]

Anabolic resistance to a mechanical stimulus may contribute to the loss of skeletal muscle mass observed with age. In this study, young and aged mice were injected with saline or human LM-111 (1 mg/kg). One week later, the myotendinous junction of the gastrocnemius muscle was removed via myotenectomy (MTE), thus placing a chronic mechanical stimulus on the remaining plantaris muscle for 2 weeks. LM-111 increased α7B integrin protein expression and clustering of the α7B integrin near DAPI+ nuclei in aged muscle in response to MTE. LM-111 reduced CD11b+ immune cells, enhanced repair, and improved the growth response to loading in aged plantaris muscle. These results suggest that LM-111 may represent a novel therapeutic approach to prevent and/or treat sarcopenia. [ABSTRACT FROM AUTHOR]

The complete 16,345-bp mitochondrial genome of the agriculturally destructive pod sucking pest, the giant coreid bug, Anoplocnemis curvipes (Hemiptera: Coreidae), was assembled from paired-end Illumina HiSeq 2500 reads. The A. curvipes mitochondrial genome consists of 13 protein coding genes (PCGs), 22 tRNAs, 2 rRNAs and a control region in the order and orientation typical among insects. PCG initiation codons (ATG, ATC, ATT and ATA) with termination codon (TAA) are used with the exception of TAG stop codons by Cytb and ND3. All tRNA genes fold into predicted cloverleaf secondary structures having requisite triplets on the anticodon loop, apart from tRNA-Ser1 (AGN) whose dihydrouridine (DHU) arm forms a simple loop. The phylogenetic analysis of hemipteran mitogenomes clusters to the family level and supports the monophyly of the five superfamilies in Pentatomomorpha of Hemiptera. The Coreoidea and Pyrrhocoroidea are sister groups, while Coreidae and Alydidae are sister groups to Rhopalidae. These analyses provide insight to mitogenomics and evolutionary relationships among pentatomoid insects. [ABSTRACT FROM AUTHOR]

Insecticide-resistant Drosophila melanogaster strains represent a resource for the discovery of the underlying molecular mechanisms of cytochrome P450 constitutive over-expression, even if some of these P450s are not directly involved in the resistance phenotype. For example, in select 4,4’-dichlorodiphenyltrichloroethane (DDT) resistant strains the glucocorticoid receptor-like (GR-like) potential transcription factor binding motifs (TFBMs) have previously been shown to be associated with constitutively differentially-expressed cytochrome P450s, Cyp12d1, Cyp6g2 and Cyp9c1. However, insects are not known to have glucocorticoids. The only ortholog to the mammalian glucocorticoid receptor (GR) in D. melanogaster is an estrogen-related receptor (ERR) gene, which has two predicted alternative splice isoforms (ERRa and ERRb). Sequencing of ERRa and ERRb in select DDT susceptible and resistant D. melanogaster strains has revealed a glycine (G) codon insertion which was only observed in the ligand binding domain of ERR from the resistant strains tested (ERR-G). Transgenic flies, expressing the ERRa-G allele, constitutively over-expressed Cyp12d1, Cyp6g2 and Cyp9c1. Only Cyp12d1 and Cyp6g2 were over-expressed in the ERRb-G transgenic flies. Phylogenetic studies show that the G-insertion appeared to be located in a less conserved domain in ERR and this insertion is found in multiple species across the Sophophora subgenera. [ABSTRACT FROM AUTHOR]

Adaptation of insect phenotypes for survival after exposure to xenobiotics can result from selection at multiple loci with additive genetic effects. To the authors’ knowledge, no selective sweep analysis has been performed to identify such loci in highly dichlorodiphenyltrichloroethane (DDT) resistant insects. Here we compared a highly DDT resistant phenotype in the Drosophila melanogaster (Drosophila) 91-R strain to the DDT susceptible 91-C strain, both of common origin. Whole genome re-sequencing data from pools of individuals was generated separately for 91-R and 91-C, and mapped to the reference Drosophila genome assembly (v. 5.72). Thirteen major and three minor effect chromosome intervals with reduced nucleotide diversity (π) were identified only in the 91-R population. Estimates of Tajima's D (D) showed corresponding evidence of directional selection in these same genome regions of 91-R, however, no similar reductions in π or D estimates were detected in 91-C. An overabundance of non-synonymous proteins coding to synonymous changes were identified in putative open reading frames associated with 91-R. Except for NinaC and Cyp4g1, none of the identified genes were the ‘usual suspects’ previously observed to be associated with DDT resistance. Additionally, up-regulated ATP-binding cassette transporters have been previously associated with DDT resistance; however, here we identified a structurally altered MDR49 candidate resistance gene. The remaining fourteen genes have not previously been shown to be associated with DDT resistance. These results suggest hitherto unknown mechanisms of DDT resistance, most of which have been overlooked in previous transcriptional studies, with some genes having orthologs in mammals. [ABSTRACT FROM AUTHOR]

The Drosophila melanogaster 91-R and 91-C strains are of common origin, however, 91-R has been intensely selected for dichlorodiphenyltrichloroethane (DDT) resistance over six decades while 91-C has been maintained as the non-selected control strain. These fly strains represent a unique genetic resource to understand the accumulation and fixation of mutations under laboratory conditions over decades of pesticide selection. Considerable research has been done to investigate the differential expression of genes associated with the highly DDT resistant strain 91-R, however, with the advent of whole genome sequencing we can now begin to develop an in depth understanding of the genomic changes associated with this intense decades-long xenobiotic selection pressure. Here we present the first whole genome sequencing analysis of the 91-R and 91-C fly strains to identify genome-wide structural changes within the open reading frames. Between-strain changes in allele frequencies revealed a higher percent of new alleles going to fixation for the 91-R strain, as compared to 91-C (P<0.0001). These results suggest that resistance to DDT in the 91-R laboratory strain could potentially be due primarily to new mutations, as well as being polygenic rather than the result of a few major mutations, two hypotheses that remain to be tested. [ABSTRACT FROM AUTHOR]

The α7β1-integrin is an adhesion molecule highly expressed in skeletal muscle that can enhance regeneration in response to eccentric exercise. We have demonstrated that mesenchymal stem cells (MSCs), predominantly pericytes, accumulate in muscle (mMSCs) overexpressing the α7B-integrin (MCK:α7B; α7Tg) and contribute to new fiber formation following exercise. Since vascularization is a common event that supports tissue remodeling, we hypothesized that the α7-integrin and/or mMSCs may stimulate vessel growth following eccentric exercise. Wild-type (WT) and α7Tg mice were subjected to single or multiple (3 times/wk, 4 wk) bouts of downhill running exercise. Additionally, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) -labeled mMSCs were intramuscularly injected into WT recipients. A subset of recipient mice were run downhill before injection to recapitulate the exercised microenvironment. While total number of CD31+ vessels declined in both WT and α7Tg muscle following a single bout of exercise, the number of larger CD31+ vessels with a visible lumen was preferentially increased in α7Tg mice following eccentric exercise training (P < 0.05). mMSC transplantation similarly increased vessel diameter and the total number of neuron-glial antigen-2 (NG2+) arterioles postexercise. Secretion of arteriogenic factors from mMSCs in response to mechanical strain, including epidermal growth factor and granulocyte macrophage-colony stimulating factor, may account for vessel remodeling. In conclusion, this study demonstrates that the α7-integrin and mMSCs contribute to increased vessel diameter size and arteriolar density in muscle in response to eccentric exercise. The information in this study has implications for the therapeutic treatment of injured muscle and disorders that result in vessel occlusion, including peripheral artery disease. [ABSTRACT FROM AUTHOR]

Eccentric, or lengthening, contractions result in injury and subsequently stimulate the activation and proliferation of satellite stem cells which are important for skeletal muscle regeneration. The discovery of alternative myogenic progenitors in skeletal muscle raises the question as to whether stem cells other than satellite cells accumulate in muscle in response to exercise and contribute to post-exercise repair and/or growth. In this study, stem cell antigen-1 (Sca-1) positive, nonhematopoetic (CD45-) cells were evaluated in wild type (WT) and α7 integrin transgenic (α7Tg) mouse muscle, which is resistant to injury yet liable to strain, 24 hr following a single bout of eccentric exercise. Sca-1+CD45- stem cells were increased 2-fold in WT muscle post-exercise. The α7 integrin regulated the presence of Sca-1+ cells, with expansion occurring in α7Tg muscle and minimal cells present in muscle lacking the α7 integrin. Sca-1+CD45- cells isolated from α7Tg muscle following exercise were characterized as mesenchymal-like stem cells (mMSCs), predominantly pericytes. In vitro multiaxial strain upregulated mMSC stem cells markers in the presence of laminin, but not gelatin, identifying a potential mechanistic basis for the accumulation of these cells in muscle following exercise. Transplantation of DiI-labeled mMSCs into WT muscle increased Pax7+ cells and facilitated formation of eMHC+DiI- fibers. This study provides the first demonstration that mMSCs rapidly appear in skeletal muscle in an α7 integrin dependent manner post-exercise, revealing an early event that may be necessary for effective repair and/or growth following exercise. The results from this study also support a role for the α7 integrin and/or mMSCs in molecular- and cellular-based therapeutic strategies that can effectively combat disuse muscle atrophy. [ABSTRACT FROM AUTHOR]

The α7β1-integrin is a heterodimeric transmembrane protein that adheres to laminin in the extracellular matrix, representing a critical link that maintains structure in skeletal muscle. In addition to preventing exercise-induced skeletal muscle injury, the α7-integrin has been proposed to act as an intrinsic mechanosensor, initiating cellular growth in response to mechanical strain. The purpose of this study was to determine the extent to which the α7-integrin regulates muscle hypertrophy following eccentric exercise. Wild-type (WT) and α7-integrin transgenic (α7Tg) mice completed a single bout of downhill running exercise (-20°, 17 m/min, 60 min), and gastrocnemius-soleus complexes were collected 1, 2, 4, and 7 days (D) postexercise (PE). Maximal isometric force was maintained and macrophage accumulation was suppressed in α7Tg muscle 1D PE. Mean fiber cross-sectional area was unaltered in WT mice but increased 40% in α7Tg mice 7D PE. In addition, a rapid and striking fivefold increase in embryonic myosin heavy chain-positive fibers appeared in α7Tg mice 2D PE. Although Pax7-positive satellite cells were increased in α7Tg muscle 1D PE, the number of nuclei per myofiber was not altered 7D PE. Phosphorylation of mammalian target of rapamycin (mTOR) was significantly elevated in α7Tg 1D PE. This study provides the first demonstration that the presence of the α7β1-integrin in skeletal muscle increases fiber hypertrophy and new fiber synthesis in the early time course following a single bout of eccentric exercise. Further studies are necessary to elucidate the precise mechanism by which the α7-integrin can enhance muscle hypertrophy following exercise. [ABSTRACT FROM AUTHOR]

Mechanical stimuli increase skeletal muscle growth in a mammalian target of rapamycin (mTOR)- and p70S6K-dependent manner. It has been proposed that costameric proteins at Z bands may sense and transfer tension to these initiators of protein translation, but few candidates have been identified. The purpose of this study was to determine whether a role exists for the α7-integrin in the activation of hypertrophic signaling and growth following eccentric exercise training. Five-week-old, wild-type (WT) and α7BX2-integrin transgenic (α7Tg) mice were randomly assigned to one of two groups: 1) sedentary (SED), or 2) exercise training (EX). Exercise training consisted of downhill running 3 sessions/wk for 4 wk (-20°, 17 m/min, 30 min). Downhill running was used to induce physiological mechanical strain. Twenty-four hours following the final training session, maximal isometric hindlimb plantar flexor force was measured. Gastrocnemius-soleus complexes were collected for further analysis of signaling changes, which included AKT, mTOR and p70S6K, and muscle growth. Despite increased p70S6K activity in WT/EX, no significant changes in cross-sectional area or force were observed in WT/EX compared with WT/SED. AKT, mTOR, and p70S6K activation was higher, and whole muscle hypertrophy, relative muscle weight, myofibrillar protein, and force were significantly elevated in α7Tg/EX compared with α7Tg/SED. A marked increase in average myofiber cross-sectional area was observed in α7Tg/EX compared with all groups. Our findings demonstrate that the α7β1-integrin sensitizes skeletal muscle to mechanical strain and subsequent growth. Thus the α7β1-integrin may represent a novel molecular therapy for the treatment of disuse muscle atrophy. [ABSTRACT FROM AUTHOR]

O-mannosylation is an important protein modification in eukaryotes that is initiated by an evolutionarily conserved family of protein O-mannosyltransferases. The first mammalian protein O-mannosyltransferase gene described was the human POMT1. Mutations in the hPOMT1 gene are responsible for Walker-Warburg syndrome (WWS), a severe recessive congenital muscular dystrophy associated with defects in neuronal migration that produce complex brain and eye abnormalities. During embryogenesis, the murine Pomt1 gene is prominently expressed in the neural tube, the developing eye, and the mesenchyme. These sites of expression correlate with those in which the main tissue alterations are observed in WWS patients. We have inactivated a Pomt1 allele by gene targeting in embryonic stem cells and produced chimeras transmitting the defect allele to offspring. Although heterozygous mice were viable and fertile, the total absence of Pomt1-/- pups in the progeny of heterozygous intercrosses indicated that this genotype is embryonic lethal. An analysis of the mutant phenotype revealed that homozygous Pomt1-/- mice suffer developmental arrest around embryonic day (E) 7.5 and die between E7.5 and E9.5. The Pomt1-/- embryos present defects in the formation of Reichert's membrane, the first basement membrane to form in the embryo. The failure of this membrane to form appears to be the result of abnormal glycosylation and maturation of dystroglycan that may impair recruitment of laminin, a structural component required for the formation of Reichert's membrane in rodents. The targeted disruption of mPomt1 represents an example of an engineered deletion of a known glycosyltransferase involved in O-mannosyl glycan synthesis. [ABSTRACT FROM AUTHOR]

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder affecting several systems caused by a heterozygous deletion in the chromosomal region 7q11.23. A common interval that includes up to 17 genes reported so far is deleted in the great majority of patients. Elastin haploinsufficiency is responsible for the cardiovascular features, but the specific contribution of other deleted genes to the WBS phenotype remains unknown. We have fully characterised a gene commonly deleted in WBS, WBSCR14, previously reported in a truncated form as WS-bHLH. The WBSCR14 cDNA encodes an 852 amino acid protein with a basic helix-loop-helix-leucine-zipper motif (bHLHZip) and a bipartite nuclear Iocalisation signal (BNLS), suggesting a function as a transcription factor. WBSCR14 is expressed as a 4.2 kb transcript predominantly in adult liver and at late stages of foetal development. The WBSCR14 locus encompasses 33 kb of genomic DNA with 17 exons. Two intragenic polymorphic dinucleotide repeats have been identified and used to verify hemizygosity in WBS patients. We have also cloned the mouse ortholog and mapped its locus to mouse chromosome 5, in a region of conserved synteny with human 7q11.23. Given that other bHLHZip proteins are dosage sensitive and based on the putative function of WBSCR14 as a transcription factor, hemizygosity at this locus could be involved in some features of WBS. [ABSTRACT FROM AUTHOR]

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder. To date, a relatively small number of NF1 mutations have been characterized, thus precluding genotype-phenotype correlations. By genotyping 75 NF1 families, we have detected six hemizygous patients (two of whom are members of the same family). The five presumed deletions were confirmed by two quantitative methods of analysis of NF1 copy number: Southern hybridization with cDNA probes and a single-strand conformation polymorphism analysis that discriminates between the NF1 gene and the pseudogene sequences. The five deletions remove most of the NF1 gene, at least 225 kb, from exon 9 to the 3′ end of the coding sequence. The origin of de novo mutations in the NF1 gene has been reported to be mainly paternal but we have determined that four of the de novo deletions involved the maternal chromosome and one the paternal chromosome. The six patients with deletions exhibited precocious, multiple clinical features of the disease. The incidence of tumor complications, particularly plexiform neurofibromas and intracranial tumors, among this group of patients is higher than the observed incidence in our NF1 population, suggesting that NF1 haploinsufficiency may cause a more severe phenotype with regard to tumor development. In contrast to other reports that associated large deletions with mildly dysmorphic facies, mental retardation and a large number of cutaneous neurofibromas, only one out of our six patients presented this phenotype. [ABSTRACT FROM AUTHOR]

The organochloride insecticide dichlorodiphenyltrichloroethane (DDT) and its metabolites can increase cellular levels of reactive oxygen species (ROS), cause mitochondrial dysfunction, and induce apoptosis. The highly DDT-resistant Drosophila melanogaster Meigen 1830 (Drosophila) strain, 91-R, and its susceptible control, 91-C, were used to investigate functional and structural changes among mitochondrial-derived pathways. Resequencing of mitochondrial genomes (mitogenomes) detected no structural differences between 91-R and 91-C, whereas RNA-seq suggested the differential expression of 221 mitochondrial-associated genes. Reverse transcriptase-quantitative PCR validation of 33 candidates confirmed that transcripts for six genes (Cyp12d1-p, Cyp12a4, cyt-c-d, COX5BL, COX7AL, CG17140) were significantly upregulated and two genes (Dif, Rel) were significantly downregulated in 91-R. Among the upregulated genes, four genes are duplicated within the reference genome (cyt-c-d, CG17140, COX5BL, and COX7AL). The predicted functions of the differentially expressed genes, or known functions of closely related genes, suggest that 91-R utilizes existing ROS regulation pathways of the mitochondria to combat increased ROS levels from exposure to DDT. This study represents, to our knowledge, the initial investigation of mitochondrial genome sequence variants and functional adaptations in responses to intense DDT selection and provides insights into potential adaptations of ROS management associated with DDT selection in Drosophila. [ABSTRACT FROM AUTHOR]