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Sample records for dystrophin exon skipping

  1. Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries

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    Fletcher Sue

    2007-07-01

    Full Text Available Abstract Background Antisense oligonucleotides (AOs can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting. Results Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers. Conclusion The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino

  2. 2′-O-Methyl RNA/Ethylene-Bridged Nucleic Acid Chimera Antisense Oligonucleotides to Induce Dystrophin Exon 45 Skipping

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    Tomoko Lee

    2017-02-01

    Full Text Available Duchenne muscular dystrophy (DMD is a fatal muscle-wasting disease characterized by dystrophin deficiency from mutations in the dystrophin gene. Antisense oligonucleotide (AO-mediated exon skipping targets restoration of the dystrophin reading frame to allow production of an internally deleted dystrophin protein with functional benefit for DMD patients who have out-of-frame deletions. After accelerated US approval of eteplirsen (Exondys 51, which targets dystrophin exon 51 for skipping, efforts are now focused on targeting other exons. For improved clinical benefits, this strategy requires more studies of the delivery method and modification of nucleic acids. We studied a nucleotide with a 2′-O,4′-C-ethylene-bridged nucleic acid (ENA, which shows high nuclease resistance and high affinity for complementary RNA strands. Here, we describe the process of developing a 2′-O-methyl RNA(2′-OMeRNA/ENA chimera AO to induce dystrophin exon 45 skipping. One 18-mer 2′-OMeRNA/ENA chimera (AO85 had the most potent activity for inducing exon 45 skipping in cultured myotubes. AO85 was administered to mdx mice without significant side effects. AO85 transfection into cultured myotubes from 13 DMD patients induced exon 45 skipping in all samples at different levels and dystrophin expression in 11 patients. These results suggest the possible efficacy of AO-mediated exon skipping changes in individual patients and highlight the 2′-OMeRNA/ENA chimera AO as a potential fundamental treatment for DMD.

  3. Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy

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    van Ommen Gert Jan B

    2011-04-01

    Full Text Available Abstract Background Myostatin is a potent muscle growth inhibitor that belongs to the Transforming Growth Factor-β (TGF-β family. Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms. By contrast, Duchenne muscular dystrophy (DMD is characterized by a loss of muscle fibers and impaired regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients. Methods We targeted myostatin exon 2 using antisense oligonucleotides (AON in healthy and DMD-derived myotubes cultures. We assessed the exon skipping level, transcriptional expression of myostatin and its target genes, and combined myostatin and several dystrophin AONs. These AONs were also applied in the mdx mice models via intramuscular injections. Results Myostatin AON induced exon 2 skipping in cell cultures and to a lower extent in the mdx mice. It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression. Furthermore, combination of myostatin and dystrophin AONs induced simultaneous skipping of both genes. Conclusions We conclude that two AONs can be used to target two different genes, MSTN and DMD, in a straightforward manner. Targeting multiple ligands of TGF-beta family will be more promising as adjuvant therapies for DMD.

  4. Deletion of Dystrophin In-Frame Exon 5 Leads to a Severe Phenotype: Guidance for Exon Skipping Strategies.

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    Zhi Yon Charles Toh

    Full Text Available Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.

  5. Targeted exon skipping to correct exon duplications in the dystrophin gene

    National Research Council Canada - National Science Library

    Greer, Kane L; Lochmüller, Hanns; Flanigan, Kevin; Fletcher, Susan; Wilton, Steve D

    2014-01-01

    .... Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide...

  6. Identification of small molecule and genetic modulators of AON-induced dystrophin exon skipping by high-throughput screening.

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    Debra A O'Leary

    Full Text Available One therapeutic approach to Duchenne Muscular Dystrophy (DMD recently entering clinical trials aims to convert DMD phenotypes to that of a milder disease variant, Becker Muscular Dystrophy (BMD, by employing antisense oligonucleotides (AONs targeting splice sites, to induce exon skipping and restore partial dystrophin function. In order to search for small molecule and genetic modulators of AON-dependent and independent exon skipping, we screened approximately 10,000 known small molecule drugs, >17,000 cDNA clones, and >2,000 kinase- targeted siRNAs against a 5.6 kb luciferase minigene construct, encompassing exon 71 to exon 73 of human dystrophin. As a result, we identified several enhancers of exon skipping, acting on both the reporter construct as well as endogenous dystrophin in mdx cells. Multiple mechanisms of action were identified, including histone deacetylase inhibition, tubulin modulation and pre-mRNA processing. Among others, the nucleolar protein NOL8 and staufen RNA binding protein homolog 2 (Stau2 were found to induce endogenous exon skipping in mdx cells in an AON-dependent fashion. An unexpected but recurrent theme observed in our screening efforts was the apparent link between the inhibition of cell cycle progression and the induction of exon skipping.

  7. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

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    HaiFang Yin

    2013-01-01

    Full Text Available We have recently reported that cell-penetrating peptides (CPPs and novel chimeric peptides containing CPP (referred as B peptide and muscle-targeting peptide (referred as MSP motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO and control peptide 3 (B-3-PMO and 3-B-PMO were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO, further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG, indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.

  8. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency.

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    Yin, Haifang; Boisguerin, Prisca; Moulton, Hong M; Betts, Corinne; Seow, Yiqi; Boutilier, Jordan; Wang, Qingsong; Walsh, Anthony; Lebleu, Bernard; Wood, Matthew Ja

    2013-09-24

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Molecular Therapy-Nucleic Acids (2013) 2, e124; doi:10.1038/mtna.2013

  9. Effective exon skipping and dystrophin restoration by 2'-o-methoxyethyl antisense oligonucleotide in dystrophin-deficient mice.

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    Lu Yang

    Full Text Available Antisense oligonucleotide (AO-mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2'-O-methoxyethyl oligonucleotides (MOE on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS AOs provided the greatest exon-skipping efficacy. When compared with 2'O methyl phosphorothioate (2'OmePS AOs, 25-mer MOE (PS AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.

  10. A case of Becker muscular dystrophy resulting from the skipping of four contiguous exons (71-74) of the dystrophin gene during mRNA maturation.

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    Patria, S Y; Alimsardjono, H; Nishio, H; Takeshima, Y; Nakamura, H; Matsuo, M

    1996-07-01

    The mutations in one-third of both Duchenne and Becker muscular dystrophy patients remain unknown because they do not involve gross rearrangements of the dystrophin gene. Here we report the first example of multiple exon skipping during the splicing of dystrophin mRNA precursor encoded by an apparently normal dystrophin gene. A 9-year-old Japanese boy exhibiting excessive fatigue and high serum creatine kinase activity was examined for dystrophinopathy. An immunohistochemical study of muscle tissue biopsy disclosed faint and discontinuous staining of the N-terminal and rod domains of dystrophin but no staining at all of the C-terminal domain of dystrophin. The dystrophin transcript from muscle tissue was analyzed by the reverse transcriptase polymerase chain reaction. An amplified product encompassing exons 67-79 of dystrophin cDNA was found to be smaller than that of the wild-type product. Sequence analysis of this fragment showed that the 3' end of exon 70 was directly connected to the 5' end of exon 75 and, thus, that exons 71-74 were completely absent. As a result, a truncated dystrophin protein lacking 110 amino acids from the C-terminal domain should result from translation of this truncated mRNA, and the patient was diagnosed as having Becker muscular dystrophy at the molecular level. Genomic DNA was analyzed to identify the cause of the disappearance of these exons. Every exon-encompassing region could be amplified from genomic DNA, indicating that the dystrophin gene is intact. Furthermore, sequencing of these amplified products did not disclose any particular nucleotide change that could be responsible for the multiple exon skipping observed. Considering that exons 71-74 are spliced out alternatively in some tissue-specific isoforms, to suppose that the alternative splicing machinery is present in the muscle tissue of the index case and that it is activated by an undetermined mechanism is reasonable. These results illustrate a novel genetic anomaly that

  11. Nanopolymers improve delivery of exon skipping oligonucleotides and concomitant dystrophin expression in skeletal muscle of mdx mice

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    Sirsi Shashank R

    2008-04-01

    Full Text Available Abstract Background Exon skipping oligonucleotides (ESOs of 2'O-Methyl (2'OMe and morpholino chemistry have been shown to restore dystrophin expression in muscle fibers from the mdx mouse, and are currently being tested in phase I clinical trials for Duchenne Muscular Dystrophy (DMD. However, ESOs remain limited in their effectiveness because of an inadequate delivery profile. Synthetic cationic copolymers of poly(ethylene imine (PEI and poly(ethylene glycol (PEG are regarded as effective agents for enhanced delivery of nucleic acids in various applications. Results We examined whether PEG-PEI copolymers can facilitate ESO-mediated dystrophin expression after intramuscular injections into tibialis anterior (TA muscles of mdx mice. We utilized a set of PEG-PEI copolymers containing 2 kDa PEI and either 550 Da or 5 kDa PEG, both of which bind 2'OMe ESOs with high affinity and form stable nanoparticulates with a relatively low surface charge. Three weekly intramuscular injections of 5 μg of ESO complexed with PEI2K-PEG550 copolymers resulted in about 500 dystrophin-positive fibers and about 12% of normal levels of dystrophin expression at 3 weeks after the initial injection, which is significantly greater than for injections of ESO alone, which are known to be almost completely ineffective. In an effort to enhance biocompatibility and cellular uptake, the PEI2K-PEG550 and PEI2K-PEG5K copolymers were functionalized by covalent conjugation with nanogold (NG or adsorbtion of colloidal gold (CG, respectively. Surprisingly, using the same injection and dosing regimen, we found no significant difference in dystrophin expression by Western blot between the NG-PEI2K-PEG550, CG-PEI2K-PEG5K, and non-functionalized PEI2K-PEG550 copolymers. Dose-response experiments using the CG-PEI2K-PEG5K copolymer with total ESO ranging from 3–60 μg yielded a maximum of about 15% dystrophin expression. Further improvements in dystrophin expression up to 20% of normal

  12. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ48-50 DMD cells

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    De Angelis, Fernanda Gabriella; Sthandier, Olga; Berarducci, Barbara; Toso, Silvia; Galluzzi, Giuliana; Ricci, Enzo; Cossu, Giulio; Bozzoni, Irene

    2002-01-01

    Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48–50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5′ and 3′ splice sites are coexpressed in the same cell. PMID:12077324

  13. A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient cavalier king charles spaniels is amenable to exon 51 skipping.

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    Gemma L Walmsley

    Full Text Available BACKGROUND: Duchenne muscular dystrophy (DMD, which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion "hot spot" is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD "hot spot". METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD. The dogs harbour a missense mutation in the 5' donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. CONCLUSIONS/SIGNIFICANCE: Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.

  14. Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.

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    Keng Boon Wee

    Full Text Available Antisense oligonucleotides (AONs mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency of 94% of 176 previously reported AONs. Four novel insights are inferred: (1 the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2 engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3 the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4 engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.

  15. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

    DEFF Research Database (Denmark)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most pr...... oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA)....

  16. A novel point mutation (G[sup [minus]1] to T) in a 5[prime] splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker Muscular Dystrophy

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    Hagiwara, Yoko; Nishio, Hisahide; Kitoh, Yoshihiko; Takeshima, Yasuhiro; Narita, Naoko; Wada, Hiroko; Yokoyama, Mitsuhiro; Nakamura, Hajime; Matsuo, Masafumi (Kobe Univ. School of Medicine (Japan))

    1994-01-01

    The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. The authors now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5[prime] splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5[prime] splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G[sup [minus]1]-to-T mutation at the 5[prime] splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection. 48 refs., 5 figs.

  17. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy

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    Alberto Malerba

    2012-01-01

    Full Text Available The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD. In DMD patients, where major muscle loss has occurred due to a lack of dystrophin, the therapeutic restoration of dystrophin expression alone in older patients may not be sufficient to restore the functionality of the muscles. We recently demonstrated that phosphorodiamidate morpholino oligomers (PMOs can be used to re-direct myostatin splicing and promote the expression of an out-of-frame transcript so reducing the amount of the synthesized myostatin protein. Furthermore, the systemic administration of the same PMO conjugated to an octaguanidine moiety (Vivo-PMO led to a significant increase in the mass of soleus muscle of treated mice. Here, we have further optimized the use of Vivo-PMO in normal mice and also tested the efficacy of the same PMO conjugated to an arginine-rich cell-penetrating peptide (B-PMO. Similar experiments conducted in mdx dystrophic mice showed that B-PMO targeting myostatin is able to significantly increase the tibialis anterior (TA muscle weight and when coadministered with a B-PMO targeting the dystrophin exon 23, it does not have a detrimental interaction. This study confirms that myostatin knockdown by exon skipping is a potential therapeutic strategy to counteract muscle wasting conditions and dual myostatin and dystrophin skipping has potential as a therapy for DMD.

  18. Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

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    van Ommen Gert-Jan B

    2007-07-01

    Full Text Available Abstract Background Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD. Using antisense oligonucleotides (AONs targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts. Methods Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed. Results For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62, by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons. Conclusion The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.

  19. Therapeutic effects of exon skipping and losartan on skeletal muscle of mdx mice.

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    Lee, Eun-Joo; Kim, Ah-Young; Lee, Eun-Mi; Lee, Myeong-Mi; Min, Chang-Woo; Kang, Kyung-Ku; Park, Jin-Kyu; Hwang, Meeyul; Kwon, Soon-Hak; Tremblay, Jacques P; Jeong, Kyu-Shik

    2014-08-01

    Various attempts have been made to find treatments for Duchenne muscular dystrophy (DMD) patients. Exon skipping is one of the promising technologies for DMD treatment by restoring dystropin protein, which is one of the muscle components. It is well known that losartan, an angiotensin II type1 receptor blocker, promotes muscle regeneration and differentiation by lowering the level of transforming growth factor-beta1 signaling. In this study, we illustrated the combined effects of exon skipping and losartan on skeletal muscle of mdx mice. We supplied mdx mice with losartan for 2 weeks before exon skipping treatment. The losartan with the exon skipping group showed less expression of myf5 than the losartan treated group. Also the losartan with exon skipping group recovered normal muscle architecture, in contrast to the losartan group which still showed many central nuclei. However, the exon skipping efficiency and the restoration of dystrophin protein were lower in the losartan with exon skipping group compared to the exon skipping group. We reveal that losartan promotes muscle regeneration and shortens the time taken to restore normal muscle structure when combined with exon skipping. However, combined treatment of exon skipping and losartan decreases the restoration of dystrophin protein meaning decrease of exon skipping efficiency.

  20. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

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    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-05-24

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented.

  1. Exon exchange approach to repair Duchenne dystrophin transcripts.

    Directory of Open Access Journals (Sweden)

    Stéphanie Lorain

    Full Text Available BACKGROUND: Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5' or the 3' part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon. METHODOLOGY/PRINCIPAL FINDINGS: As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23. This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25-45% of repair depending on the construction in use. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations.

  2. Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients.

    NARCIS (Netherlands)

    Helderman-van den Enden, A.T.; Straathof, C.S.; Aartsma-Rus, A.; Dunnen, J.T. den; Verbist, B.M.; Bakker, E.; Verschuuren, J.J.; Ginjaar, H.B.

    2010-01-01

    Theoretically, 13% of patients with Duchenne muscular dystrophy may benefit from antisense-mediated skipping of exon 51 to restore the reading frame, which results in the production of a shortened dystrophin protein. We give a detailed description with longitudinal follow up of three patients with B

  3. Chemical and mechanistic toxicology evaluation of exon skipping phosphorodiamidate morpholino oligomers in mdx mice.

    Science.gov (United States)

    Sazani, Peter; Ness, Kirk P Van; Weller, Doreen L; Poage, Duane; Nelson, Keith; Shrewsbury, And Stephen B

    2011-05-01

    AVI-4658 is a phosphorodiamidate morpholino oligomer (PMO) designed to induce skipping of dystrophin exon 51 and restore its expression in patients with Duchenne muscular dystrophy (DMD). Preclinically, restoration of dystrophin in the dystrophic mdx mouse model requires skipping of exon 23, achieved with the mouse-specific PMO, AVI-4225. Herein, we report the potential toxicological consequences of exon skipping and dystrophin restoration in mdx mice using AVI-4225. We also evaluated the toxicological effects of AVI-4658 in both mdx and wild-type mice. In both studies, animals were dosed once weekly for 12 weeks up to the maximum feasible dose of 960 mg/kg per injection. Both AVI-4658 and AVI-4225 were well-tolerated at all doses. Findings in AVI-4225-treated animals were generally limited to mild renal tubular basophilia/vacuolation, without any significant changes in renal function and with evidence of reversing. No toxicity associated with the mechanism of action of AVI-4225 in a dystrophic animal was observed.

  4. Novel Cationic Carotenoid Lipids as Delivery Vectors of Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Vassilia Partali

    2012-01-01

    Full Text Available Duchenne Muscular Dystrophy (DMD is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs. The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC.

  5. Detection of an exon 53 polymorphism in the dystrophin gene.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Sedra, M S

    1993-10-01

    We utilized a heteroduplex method to screen for small mutations in Duchenne muscular dystrophy patients who did not have deletions or duplications. A dystrophin exon 53 heteroduplex band was identified in 14.4% of the affected patients. Direct sequencing of the amplified product from DNA producing the heteroduplex revealed the presence of a polymorphism in the coding region. The codon for asparagine was converted from AAT to AAC.

  6. Wild-type mouse models to screen antisense oligonucleotides for exon-skipping efficacy in Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Limin Cao

    Full Text Available A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD, a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.

  7. Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

    Directory of Open Access Journals (Sweden)

    Christa L Tanganyika-de Winter

    2012-01-01

    Full Text Available Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs. Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype. Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

  8. Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy

    Science.gov (United States)

    Verheul, Ruurd C.; van Deutekom, Judith C. T.; Datson, Nicole A.

    2016-01-01

    Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0–100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD. PMID:27612288

  9. Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

    Science.gov (United States)

    Jirka, Silvana M G; Heemskerk, Hans; Tanganyika-de Winter, Christa L; Muilwijk, Daan; Pang, Kar Him; de Visser, Peter C; Janson, Anneke; Karnaoukh, Tatyana G; Vermue, Rick; 't Hoen, Peter A C; van Deutekom, Judith C T; Aguilera, Begoña; Aartsma-Rus, Annemieke

    2014-02-01

    Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.

  10. Translational and Regulatory Challenges for Exon Skipping Therapies

    NARCIS (Netherlands)

    Aartsma-Rus, Annemieke; Ferlini, Alessandra; Goemans, Nathalie; Pasmooij, Anna M. G.; Wells, Dominic J.; Bushby, Katerine; Vroom, Elizabeth; Balabanov, Pavel

    2014-01-01

    Several translational challenges are currently impeding the therapeutic development of antisense-mediated exon skipping approaches for rare diseases. Some of these are inherent to developing therapies for rare diseases, such as small patient numbers and limited information on natural history and int

  11. Antisense-mediated exon skipping to reframe transcripts.

    Science.gov (United States)

    Turczynski, Sandrina; Titeux, Matthias; Pironon, Nathalie; Hovnanian, Alain

    2012-01-01

    Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.

  12. Variants affecting exon skipping contribute to complex traits.

    Directory of Open Access Journals (Sweden)

    Younghee Lee

    Full Text Available DNA variants that affect alternative splicing and the relative quantities of different gene transcripts have been shown to be risk alleles for some Mendelian diseases. However, for complex traits characterized by a low odds ratio for any single contributing variant, very few studies have investigated the contribution of splicing variants. The overarching goal of this study is to discover and characterize the role that variants affecting alternative splicing may play in the genetic etiology of complex traits, which include a significant number of the common human diseases. Specifically, we hypothesize that single nucleotide polymorphisms (SNPs in splicing regulatory elements can be characterized in silico to identify variants affecting splicing, and that these variants may contribute to the etiology of complex diseases as well as the inter-individual variability in the ratios of alternative transcripts. We leverage high-throughput expression profiling to 1 experimentally validate our in silico predictions of skipped exons and 2 characterize the molecular role of intronic genetic variations in alternative splicing events in the context of complex human traits and diseases. We propose that intronic SNPs play a role as genetic regulators within splicing regulatory elements and show that their associated exon skipping events can affect protein domains and structure. We find that SNPs we would predict to affect exon skipping are enriched among the set of SNPs reported to be associated with complex human traits.

  13. Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells.

    Science.gov (United States)

    Lehto, Taavi; Castillo Alvarez, Alejandra; Gauck, Sarah; Gait, Michael J; Coursindel, Thibault; Wood, Matthew J A; Lebleu, Bernard; Boisguerin, Prisca

    2014-03-01

    Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.

  14. Polyquaternium-mediated delivery of morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

    Science.gov (United States)

    Wang, Mingxing; Wu, Bo; Shah, Sapana N; Lu, Peijuan; Lu, Qilong

    2017-11-01

    Antisense oligonucleotide therapy for Duchenne muscular dystrophy has shown great potential in preclinical and clinical trials, but its therapeutic applications are still limited due to inefficient delivery. In this study, we investigated a few polyquaterniums (PQs) with different size and composition for their potential to improve delivery performance of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that Luviquat(TM) series, especially PQ-1 and PQ-3, promoted the exon-skipping efficiency comparable to Endoporter-mediated PMO delivery in vitro. Significant enhancement in skipping dystrophin exon 23 has also been achieved with PQ-3 up to seven-fold when compared to PMO alone in mdx mice. Cytotoxicity of the PQs was lower than Endoporter and PEI 25 K in vitro and muscle damage not clearly detected in vivo under the tested concentrations. These results together demonstrate that the optimization of PQ in molecular size, composition and distribution of positive charges is the key factor to achieve enhanced PMO exon-skipping efficiency. The higher efficiency and lower toxicity endow polyquaternium series as AO delivery enhancing agents for treating muscular dystrophy and other diseases.

  15. Exon skipping and Duchenne muscular dystrophy: Hope, hype and how feasible?

    Directory of Open Access Journals (Sweden)

    Wilton Steve

    2008-01-01

    Full Text Available Duchenne muscular dystrophy (DMD, the most common and serious form of childhood muscle wasting is generally caused by protein-truncating mutations in the large DMD gene. Specific removal of an exon from a defective DMD gene transcript has the potential to allow synthesis of a semi-functional dystrophin, thereby reducing the severity and presumably progression of muscle wasting. The efficacy of this treatment will vary greatly between the different mutations that preclude the synthesis of a functional dystrophin. Restoration of the reading frame from a large multi-exon genomic deletion, typically greater than 36 exons, may lead to synthesis of a protein with only partial function and limited clinical benefit, whereas excising a nonsense mutation in a redundant exon should generate a near normal dystrophin. A clinical trial has recently confirmed proof-of-principle that exclusion of Exon 51 from human dystrophin mRNAs, carrying frame-shifting deletions adjacent to this exon, results in dystrophin expression. No major side-effects after local administration of the antisense oligomer were reported. Additional trials are underway, targeting the same exon but using an oligomer of different backbone chemistry. If functional dystrophin synthesis is demonstrated, and safety issues are addressed, subsequent trials will involve systemic delivery. Great challenges are ahead, some technical; establishing an effective delivery regimen, some ethical; choosing subsequent targets for therapy, and others of an administrative and regulatory nature.

  16. Evidence for association of multi-exon skipping events with tumors

    Institute of Scientific and Technical Information of China (English)

    Jianning BI; Tao PENG; Yanda LI

    2008-01-01

    Alternative splicing(AS)has been shown to be frequently present in human tumors.Specifically,it has been observed in some experimental studies that multi-exon skipping(MES)events often appear in tumorous tissues.Prompted by this observation,we conducted a genomewide analysis of MES events to investigate their association with tumors.The results show that MES events are more likely associated with tumors than single-exon skipping (SES) and the degree of association increases with the number of skipped exons.Furthermore,MES events are found to be less conserved than their SES counterparts,which provides additional evidence for our results because disease-associated AS events should be eliminated during evolution.Interestingly,these differences still existed even after comparison Of MES and SES events with similarlength skipped regions.These results demonstrate that MES events mav be associated with tumors and suggest that MES isoforms might be useful in cancer diagnosis.

  17. Monoclonal antibodies against the muscle-specific N-terminus of dystrophin: Characterization of dystrophin in a muscular dystrophy patient with a frameshift deletion of Exons 3-7

    Energy Technology Data Exchange (ETDEWEB)

    Thanh, L. T.; Man, N. thi; Morris, G.E. (North East Wales Institute, Clwyd (United Kingdom)); Love, D.R.; Davies, K.E. (Institute of Molecular Medicine, John Radcliffe Hospital, Oxford (United Kingdom)); Helliwell, T.R. (Liverpool Univ. (United Kingdom))

    1993-07-01

    The first three exons of the human muscle dystrophin gene were expressed as a [beta]-galactosidase fusion protein. 1-his protein was then used to prepare two monoclonal antibodies (mAbs) which react with native dystrophin on frozen muscle sections and with denatured dystrophin on western blots but which do not cross-react with the distrophin-related protein, utrophin. Both mAbs recognized dystrophin in muscular dystrophy (MD) patients with deletions of exon 3, and further mapping with 11 overlapping synthetic peptides showed that they both recognize an epitope encoded by the muscle-specific exon 1. Neither mAb recognizes the brain dystrophin isoform, confirming the prediction from mRNA data that this has a different N-terminus. One Becker MD patient with a frameshift deletion of exons 3-7 is shown to produce dystrophin which reacts with the N-terminal mAbs, as well as with mAbs which bind on the C-terminal side of the deletion. The data suggest that transcription begins at the normal muscle dystrophin promoter and that the normal reading frame is restored after the deletion. A number of mechanisms have been proposed for restoration of the reading frame after deletion of exons 3-7, but those which predict dystrophin with an abnormal N-terminus do not appear to be major mechanisms in this patient. 27 refs., 6 figs.

  18. Cationic polyelectrolyte-mediated delivery of antisense morpholino oligonucleotides for exon-skipping in vitro and in mdx mice.

    Science.gov (United States)

    Wang, Mingxing; Wu, Bo; Tucker, Jason D; Lu, Peijuan; Lu, Qilong

    2015-01-01

    In this study, we investigated a series of cationic polyelectrolytes (PEs) with different size and composition for their potential to improve delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. The results showed that the poly(diallyldimethylammonium chloride) (PDDAC) polymer series, especially PE-3 and PE-4, improves the delivery efficiency of PMO, comparable with Endoporter-mediated PMO delivery in vitro. The enhanced PMO delivery and targeting to dystrophin exon 23 was further observed in mdx mice, up to fourfold with the PE-4, compared with PMO alone. The cytotoxicity of the PEs was lower than that of Endoporter and polyethylenimine 25,000 Da in vitro, and was not clearly detected in muscle in vivo under the tested concentrations. Together, these results demonstrate that optimization of PE molecular size, composition, and distribution of cationic charge are key factors to achieve enhanced PMO exon-skipping efficiency. The increased efficiency and lower toxicity show this PDDAC series to be capable gene/antisense oligonucleotide delivery-enhancing agents for treating muscular dystrophy and other diseases.

  19. Modulating Calcium Signals to Boost AON Exon Skipping for DMD

    Science.gov (United States)

    2016-10-01

    RyR antags for RNASeQ (18 months; 70% complete) We are in the process of prioritizing based on initial findings. As of now CDMD1003 exon 45...sequence analysis Subtask 1 - Optimize alternate splicing assay using exon capture and RNASeq (12 months, 80% complete). Additionally, we have...begun optimizing the exon capture and performed preliminary RNASeq experiments as described using exon capture. Subtask 2 - High depth RNASeQ on

  20. Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

    Science.gov (United States)

    Rutten, Julie W; Dauwerse, Hans G; Peters, Dorien J M; Goldfarb, Andrew; Venselaar, Hanka; Haffner, Christof; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M; Lesnik Oberstein, Saskia A J

    2016-04-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations

  1. Becker Muscular Dystrophy (BMD) caused by duplication of exons 3-6 of the dystrophin gene presenting as dilated cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, A.C.; Allingham-Hawkins, D.J.; Becker, L. [Univ. of Toronto, Ontario (Canada)] [and others

    1994-09-01

    X-linked dilated cardiomyopathy (XLCM) is a progressive myocardial disease presenting with congestive heart failure in teenage males without clinical signs of skeletal myopathy. Tight linkage of XLCM to the DMD locus has been demonstrated; it has been suggested that, at least in some families, XLCM is a {open_quotes}dystrophinopathy.{close_quotes} We report a 14-year-old boy who presented with acute heart failure due to dilated cardiomyopathy. He had no history of muscle weakness, but physical examination revealed pseudohypertrophy of the calf muscles. He subsequently received a heart transplantation. Family history was negative. Serum CK level at the time of diagnosis was 10,416. Myocardial biopsy showed no evidence of carditis. Dystrophin staining of cardiac and skeletal muscle with anti-sera to COOH and NH{sub 2}termini showed a patchy distribution of positivity suggestive of Becker muscular dystrophy. Analysis of 18 of the 79 dystrophin exons detected a duplication that included exons 3-6. The proband`s mother has an elevated serum CK and was confirmed to be a carrier of the same duplication. A mutation in the muscle promotor region of the dystrophin gene has been implicated in the etiology of SLCM. However, Towbin et al. (1991) argued that other 5{prime} mutations in the dystrophin gene could cause selective cardiomyopathy. The findings in our patient support the latter hypothesis. This suggests that there are multiple regions in the dystrophin gene which, when disrupted, can cause isolated dilated cardiomyopathy.

  2. Selection preserves Ubiquitin Specific Protease 4 alternative exon skipping in therian mammals.

    Science.gov (United States)

    Vlasschaert, Caitlyn; Xia, Xuhua; Gray, Douglas A

    2016-02-02

    Ubiquitin specific protease 4 (USP4) is a highly networked deubiquitinating enzyme with reported roles in cancer, innate immunity and RNA splicing. In mammals it has two dominant isoforms arising from inclusion or skipping of exon 7 (E7). We evaluated two plausible mechanisms for the generation of these isoforms: (A) E7 skipping due to a long upstream intron and (B) E7 skipping due to inefficient 5' splice sites (5'SS) and/or branchpoint sites (BPS). We then assessed whether E7 alternative splicing is maintained by selective pressure or arose from genetic drift. Both transcript variants were generated from a USP4-E7 minigene construct with short flanking introns, an observation consistent with the second mechanism whereby differential splice signal strengths are the basis of E7 skipping. Optimization of the downstream 5'SS eliminated E7 skipping. Experimental validation of the correlation between 5'SS identity and exon skipping in vertebrates pinpointed the +6 site as the key splicing determinant. Therian mammals invariably display a 5'SS configuration favouring alternative splicing and the resulting isoforms have distinct subcellular localizations. We conclude that alternative splicing of mammalian USP4 is under selective maintenance and that long and short USP4 isoforms may target substrates in various cellular compartments.

  3. The silent mutation nucleotide 744 G --> A, Lys172Lys, in exon 6 of BRCA2 results in exon skipping

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Steffensen, Ane Y; Jønson, Lars

    2009-01-01

    Germ-line mutations in BRCA2 predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent...... and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G --> A/c.516 G --> A, Lys172Lys) in exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon trapping analysis showed that the mutation results in skipping...... of exon 6 and/or both exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation....

  4. Refinement of antisense oligonucleotide mediated exon skipping as therapy for Duchenne muscular dystrophy

    NARCIS (Netherlands)

    Heemskerk, Johannes Antonius

    2011-01-01

    In recent years, modulation of mRNA has emerged as a promising therapeutic tool. For instance, in the field of neuromuscular disorders therapeutic strategies are being developed for several diseases, including antisense oligonucleotide (AON) mediated exon skipping for Duchenne Muscular Dystrophy (DM

  5. Refinement of antisense oligonucleotide mediated exon skipping as therapy for Duchenne muscular dystrophy

    NARCIS (Netherlands)

    Heemskerk, Johannes Antonius

    2011-01-01

    In recent years, modulation of mRNA has emerged as a promising therapeutic tool. For instance, in the field of neuromuscular disorders therapeutic strategies are being developed for several diseases, including antisense oligonucleotide (AON) mediated exon skipping for Duchenne Muscular Dystrophy (DM

  6. Identification of two point mutations and a one base deletion in exon 19 of the dystrophin gene by heteroduplex formation.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartello, C; Mendell, J R

    1993-03-01

    Two thirds of the Duchenne muscular dystrophy population have either gene deletions or duplications. The nondeletion/duplication cases are most likely the result of point mutations or small deletions and duplications that cannot be easily identified by current strategies. The major obstacle in identifying small mutations is due to the large size of the dystrophin gene. We selectively screened 5 DMD exons containing CpG dinucleotides in 110 DMD patients without detectable deletions or duplications. Nonsenses mutations are frequently due to a C- to -T transition within a CG dinucleotide pair. To screen for the nonsense mutations, we used the heteroduplex method. Utilizing this approach, we identified 2 different nonsense mutations and a single base deletion all occurring in exon 19. This is the first report of a clustering of small mutations in the dystrophin gene.

  7. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3.

    Science.gov (United States)

    Toonen, Lodewijk J A; Schmidt, Iris; Luijsterburg, Martijn S; van Attikum, Haico; van Roon-Mom, Willeke M C

    2016-10-12

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.

  8. Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

    Science.gov (United States)

    Suzuki, Hitoshi; Aoki, Yoshitsugu; Kameyama, Toshiki; Saito, Takashi; Masuda, Satoru; Tanihata, Jun; Nagata, Tetsuya; Mayeda, Akila; Takeda, Shin’ichi; Tsukahara, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing. PMID:27754374

  9. Screening the dystrophin gene suggests a high rate of polymorphism in general but no exonic deletions in schizophrenics

    Energy Technology Data Exchange (ETDEWEB)

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N. [Mayo Clinic/Foundation, Rochester, MN (United States)] [and others

    1994-03-15

    The dystrophin gene, located at chromosome Xp21, was evaluated as a candidate gene in chronic schizophrenia in response to the report of a large family in which schizophrenia cosegregated with Becker muscular dystrophy. Genomic DNA from 94 men with chronic schizophrenia was evaluated by Southern blot analysis using cDNA probes that span exons 1-59. No exonic deletions were identified. An unexpectedly high rate of polymorphism was calculated in this study and two novel polymorphisms were found, demonstrating the usefulness of the candidate gene approach even when results of the original study are negative. 41 refs., 1 fig., 4 tabs.

  10. Dystrophin expression in a Duchenne muscular dystrophy patient with a frame shift deletion.

    Science.gov (United States)

    Prior, T W; Bartolo, C; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Kissel, J T; Luquette, M H; Tsao, C Y; Mendell, J R

    1997-02-01

    The exon 45 deletion is a common dystrophin gene deletion. Although this is an out-of-frame deletion, which should not allow for protein synthesis, it has been observed in mildly affected patients. We describe a patient with an exon 45 deletion who produced protein, but still had a severe Duchenne muscular dystrophy phenotype. RT-PCR analysis and cDNA sequencing from the muscle biopsy sample revealed that the exon 45 deletion induced exon skipping of exon 44, which resulted in an in-frame deletion and the production of dystrophin. A conformational change in dystrophin induced by the deletion is proposed as being responsible for the severe phenotype in the patient. We feel that the variable clinical phenotype observed in patients with the exon 45 deletion is not due to exon splicing but may be the result of other environmental or genetic factors, or both.

  11. TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML.

    Science.gov (United States)

    Mohamed, Aminetou Mint; Balsat, Marie; Koering, Catherine; Maucort-Boulch, Delphine; Boissel, Nicolas; Payen-Gay, Lea; Cheok, Meyling; Mortada, Hussein; Auboeuf, Didier; Pinatel, Christiane; El-Hamri, Mohamed; Tigaud, Isabelle; Hayette, Sandrine; Dumontet, Charles; Cros, Emeline; Flandrin-Gresta, Pascale; Nibourel, Olivier; Preudhomme, Claude; Thomas, Xavier; Nicolini, Franck-Emmanuel; Solly, Françoise; Guyotat, Denis; Campos, Lydia; Michallet, Mauricette; Ceraulo, Antony; Mortreux, Franck; Wattel, Eric

    2017-01-16

    In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2S(high) patients were found to be younger than TET2(low) patients without a difference in the rate of complete remission. However, TET2E2S(high) patients exhibited a significantly lower CIR (p<10(-4)). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

  12. Identification of a novel first exon in the human dystrophin gene and of a new promoter located more than 500 kb upstream of the nearest known promoter

    Energy Technology Data Exchange (ETDEWEB)

    Yanagawa, H.; Nishio, H.; Takeshima, Y. [Kobe Univ. School of Medicine (Japan)] [and others

    1994-09-01

    The dystrophin gene, which is muted in patients with Duchenne and Becker muscular dystrophies, is the largest known human gene. Five alternative promoters have been characterized until now. Here we show that a novel dystrophin isoform with a different first exon can be produced through transcription initiation at a previously-unidentified alternative promoter. The case study presented is that of patient with Duchenne muscular dystrophy who had a deletion extending from 5{prime} end of the dystrophin gene to exon 2, including all promoters previously mapped in the 5{prime} part of the gene. Transcripts from lymphoblastoid cells were found to contain sequences corresponding to exon 3, indicating the presence of new promoter upstream of this exon. The nucleotide sequence of amplified cDNA corresponding to the 5{prime} end of the new transcript indicated that the 5{prime} end of exon 3 was extended by 9 codons, only the last (most 3{prime}) of which codes for methionine. The genomic nucleotide sequence upstream from the new exon, as determined using inverse polymerase chain reaction, revealed the presence of sequences similar to a TATA box, an octamer motif and an MEF-2 element. The identified promoter/exon did not map to intron 2, as might have been expected, but to a position more than 500 kb upstream of the most 5{prime} of the previously-identified promoters, thereby adding 500 kb to the dystrophin gene. The sequence of part of the new promoter region is very similar to that of certain medium reiteration frequency repetitive sequences. These findings may help us understand the molecular evolution of the dystrophin gene.

  13. Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping.

    Science.gov (United States)

    Hawa, N S; Cockerill, F J; Vadher, S; Hewison, M; Rut, A R; Pike, J W; O'Riordan, J L; Farrow, S M

    1996-07-01

    Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disorder resulting in target organ resistance to the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). In many cases, this disorder has been shown to be due to mutations in the gene encoding vitamin D receptors (VDR). In a patient with characteristic features of this disorder, we investigated the functional defect and sequenced the coding region of the gene for mutations. Skin fibroblasts from patient and control were used to measure binding of 1,25(OH)2D3 and functional responses to the hormone. These cells were also used to prepare RNA from which cDNA was prepared and sequenced. Furthermore, genomic DNA was prepared from the fibroblasts and the intron/exon boundaries sequenced. A child with classic features of HVDRR with alopecia diagnosed as having rickets due to resistance to 1,25(OH)2D3. Nuclear association of 1,25(OH)2D3 was determined in patient and control cells and the functional response to 1,25(OH)2D3 was assessed by measurement of 25-hydroxyvitamin D-24-hydroxylase(24-hydroxylase) activity. VDR cDNA and genomic DNA prepared from patient and control cells were sequenced. Cells from the patient with HVDRR had undetectable amounts of VDR compared to control cells and did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)2D3. Sequencing of the VDR coding region after RT-PCR of RNA revealed an absence of exon 4 in patient RNA which was not due to a deletion in genomic DNA but was caused by exon skipping during RNA processing. In addition, the deletion of exon 4 sequences from RNA leads to a frameshift in translation resulting in a premature stop codon. Amplification of genomic DNA around the intron/exon boundary of exon 4 revealed a point mutation in the 5' donor splice site of intron 4. In this study, we have identified a novel mutation in the gene for vitamin D receptors in a patient with the characteristic phenotype of hereditary vitamin D resistant

  14. Early cardiac failure in a child with Becker muscular dystrophy is due to an abnormally low amount of dystrophin transcript lacking exon 13.

    Science.gov (United States)

    Ishigaki, C; Patria, S Y; Nishio, H; Yoshioka, A; Matsuo, M

    1997-12-01

    Two Japanese brothers with Becker muscular dystrophy were shown by polymerase chain reaction (PCR) and cDNA sequence analysis to produce a dystrophin gene transcript lacking a single exon: that is, number 13. Despite having the same deletion mutation, the brothers showed clearly different clinical phenotypes: the younger brother developed cardiac failure at the age of nine, while the elder brother was asymptomatic. As alternative splicing was not responsible for this clinical difference, the amount of dystrophin transcript was examined by using reverse transcription semi-nested and parallel PCR. The results showed that the amount of the dystrophin transcript in the younger brother was 20% of that of the elder brother. This finding suggested that lesser amount of dystrophin transcript in the younger brother was responsible for the early onset of cardiac failure. This would represent a novel molecular mechanism for dystrophinopathy.

  15. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    calf hypertrophy was noted. Creatine kinase was normal or raised maximally to 500 U/l. The muscle biopsy was myopathic with increased fiber size variation and many internal nuclei, but no dystrophy. No comorbidity was found. In both cases, western blot showed a reduced dystrophin band. Genetic...... associated with an exon 26 deletion. The proband, a 23-year-old man, had slightly delayed motor milestones, walking 1 1/2 years old. He had no complaints of muscle weakness, but had muscle pain. Clinical examination revealed no muscle wasting or loss of power, but his CK was 1500-7000 U/l. Muscle biopsy...... showed dystrophic changes. He had comorbidity with dystonia, slight mental retardation, low stature and neuropathy. The brother of the proband's mother came to medical attention when he was 43 years old. He complained about muscle pain. On examination, a MRC grade 4+ hip extention palsy and a discrete...

  16. Deletion of exon 26 of the dystrophin gene is associated with a mild Becker muscular dystrophy phenotype

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, Morten; Vissing, John

    2011-01-01

    calf hypertrophy was noted. Creatine kinase was normal or raised maximally to 500 U/l. The muscle biopsy was myopathic with increased fiber size variation and many internal nuclei, but no dystrophy. No comorbidity was found. In both cases, western blot showed a reduced dystrophin band. Genetic...... associated with an exon 26 deletion. The proband, a 23-year-old man, had slightly delayed motor milestones, walking 1 1/2 years old. He had no complaints of muscle weakness, but had muscle pain. Clinical examination revealed no muscle wasting or loss of power, but his CK was 1500-7000 U/l. Muscle biopsy...... showed dystrophic changes. He had comorbidity with dystonia, slight mental retardation, low stature and neuropathy. The brother of the proband's mother came to medical attention when he was 43 years old. He complained about muscle pain. On examination, a MRC grade 4+ hip extention palsy and a discrete...

  17. Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

    Directory of Open Access Journals (Sweden)

    Kole Ryszard

    2011-10-01

    Full Text Available Abstract Background Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Methods Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. Results It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. Conclusion This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

  18. AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation

    Directory of Open Access Journals (Sweden)

    Xavier Gerard

    2012-01-01

    Full Text Available Leber congenital amaurosis (LCA is a severe hereditary retinal dystrophy responsible for congenital or early-onset blindness. The most common disease-causing mutation (>10% is located deep in intron 26 of the CEP290 gene (c.2991+1655A>G. It creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. In the present study, we show that the use of antisense oligonucleotides (AONs allow an efficient skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients. These data support the feasibility of an AON-mediated exon skipping strategy to correct the aberrant splicing.

  19. TNF-α-Induced microRNAs Control Dystrophin Expression in Becker Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Alyson A. Fiorillo

    2015-09-01

    Full Text Available The amount and distribution of dystrophin protein in myofibers and muscle is highly variable in Becker muscular dystrophy and in exon-skipping trials for Duchenne muscular dystrophy. Here, we investigate a molecular basis for this variability. In muscle from Becker patients sharing the same exon 45–47 in-frame deletion, dystrophin levels negatively correlate with microRNAs predicted to target dystrophin. Seven microRNAs inhibit dystrophin expression in vitro, and three are validated in vivo (miR-146b/miR-374a/miR-31. microRNAs are expressed in dystrophic myofibers and increase with age and disease severity. In exon-skipping-treated mdx mice, microRNAs are significantly higher in muscles with low dystrophin rescue. TNF-α increases microRNA levels in vitro whereas NFκB inhibition blocks this in vitro and in vivo. Collectively, these data show that microRNAs contribute to variable dystrophin levels in muscular dystrophy. Our findings suggest a model where chronic inflammation in distinct microenvironments induces pathological microRNAs, initiating a self-sustaining feedback loop that exacerbates disease progression.

  20. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

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    Takashi Saito

    Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  1. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

    Science.gov (United States)

    Saito, Takashi; Nakamura, Akinori; Aoki, Yoshitsugu; Yokota, Toshifumi; Okada, Takashi; Osawa, Makiko; Takeda, Shin'ichi

    2010-08-18

    Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J)) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. We converted fibroblasts of CXMD(J) and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J) and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  2. Aminoacylase I deficiency due to ACY1 mRNA exon skipping.

    Science.gov (United States)

    Ferri, L; Funghini, S; Fioravanti, A; Biondi, E G; la Marca, G; Guerrini, R; Donati, M A; Morrone, A

    2014-10-01

    Aminoacylase 1 (ACY1) deficiency is a rare inborn error of metabolism of which less than 20 observations have been described. Patients exhibit urinary excretion of specific N-acetyl amino acids and manifest a heterogeneous clinical spectrum including intellectual disability, motor delay, seizures, moderate to severe mental retardation, absent speech, growth delay, muscular hypotonia and autistic features. Here, we report the case of ACY1 enzyme deficiency in a 6-year-old girl presenting severe intellectual disability, motor retardation, absence of spontaneous locomotor activity and severe speech delay. Urinary excretion of N-acetylated amino acids was present. Mutational analysis of ACY1 gene identified the new homozygous c.1001_1001+5del6 mutation, which alters the mRNA transcription leading to exon 13 skipping and inclusion of a premature stop codon (p.Lys308Glufs*7). A quantitative fluorescent multiplex-polymerase chain reaction (QFM-PCR) assay has been set up and confirmed homozygosity of the mutation in the patient's DNA. Biochemical analysis showed absence of ACY1 enzyme activity in the patient's fibroblasts. The structure of the mutated protein has been defined by homology modeling (HM). Our data endorse the hypothesis of a link between this inborn error of metabolism and the neurological manifestations observed in patients with ACY1 deficiency. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. De novo exonic mutation in MYH7 gene leading to exon skipping in a patient with early onset muscular weakness and fiber-type disproportion.

    Science.gov (United States)

    Pajusalu, Sander; Talvik, Inga; Noormets, Klari; Talvik, Tiina; Põder, Haide; Joost, Kairit; Puusepp, Sanna; Piirsoo, Andres; Stenzel, Werner; Goebel, Hans H; Nikopensius, Tiit; Annilo, Tarmo; Nõukas, Margit; Metspalu, Andres; Õunap, Katrin; Reimand, Tiia

    2016-03-01

    Here we report on a case of MYH7-related myopathy in a boy with early onset of muscular weakness and delayed motor development in infancy. His most affected muscles were neck extensors showing a dropped head sign, proximal muscles of lower limbs with positive Gower's sign, and trunk muscles. Brain and spinal cord MRI scans, echocardiography, and laboratory analyses including creatine kinase and lactate did not reveal any abnormalities. Muscle histopathology showed fiber-type disproportion. Whole exome sequencing of the parents-offspring trio revealed a novel de novo c.5655G>A p.(Ala1885=) synonymous substitution of the last nucleotide in exon 38 of the MYH7 gene. Further RNA investigations proved the skipping of exon 38 (p.1854_1885del). This is a first report of an exon-skipping mutation in the MYH7 gene causing myopathy. This report broadens both the phenotypic and genotypic spectra of MYH7-related myopathies. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Synthesis of a Morpholino Nucleic Acid (MNA-Uridine Phosphoramidite, and Exon Skipping Using MNA/2′-O-Methyl Mixmer Antisense Oligonucleotide

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    Suxiang Chen

    2016-11-01

    Full Text Available In this study, we synthesised a morpholino nucleoside-uridine (MNA-U phosphoramidite and evaluated the potential of a MNA-modified antisense oligonucleotide (AO sequences to induce exon 23 skipping in mdx mouse myotubes in vitro towards extending the applicability of morpholino chemistry with other nucleotide monomers. We designed, synthesised, and compared exon skipping efficiencies of 20 mer MNA-modified 2′-O-methyl RNA mixmer AO on a phosphorothioate backbone (MNA/2′-OMePS to the corresponding fully modified 2′-O-methyl RNA AO (2′-OMePS as a control. Our results showed that the MNA/2′-OMePS efficiently induced exon 23 skipping. As expected, the 2′-OMePS AO control yielded efficient exon 23 skipping. Under the applied conditions, both the AOs showed minor products corresponding to exon 22/23 dual exon skipping in low yield. As these are very preliminary data, more detailed studies are necessary; however, based on the preliminary results, MNA nucleotides might be useful in constructing antisense oligonucleotides.

  5. A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

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    Zlatko Radev

    Full Text Available Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD and Bethlem myopathy (BM remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

  6. A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Fumio; Awata, Hisataka; Matsuda, Ichiro [Kumamoto Univ. (Japan)

    1995-01-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this train together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

  7. A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III.

    Science.gov (United States)

    Endo, F; Awata, H; Katoh, H; Matsuda, I

    1995-01-01

    4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this strain together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

  8. Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice.

    Science.gov (United States)

    Gedicke-Hornung, Christina; Behrens-Gawlik, Verena; Reischmann, Silke; Geertz, Birgit; Stimpel, Doreen; Weinberger, Florian; Schlossarek, Saskia; Précigout, Guillaume; Braren, Ingke; Eschenhagen, Thomas; Mearini, Giulia; Lorain, Stéphanie; Voit, Thomas; Dreyfus, Patrick A; Garcia, Luis; Carrier, Lucie

    2013-07-01

    Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.

  9. Screening Duchenne and Becker muscular dystrophy patients for deletions in 30 exons of the dystrophin gene by three-multiplex PCR

    Energy Technology Data Exchange (ETDEWEB)

    Risch, N. (Yale Univ., New Haven, CT (United States))

    1992-09-01

    Deletion mutations of the dystrophin gene may cause either the severe Duchenne muscular dystrophy (DMD) or the milder, allelic Becker muscular dystrophy (BMD) and are clustered in two high-frequency-deletion regions (HFDRs) located, respectively, 500 kb and 1,200 kb downstream from the 5[prime] end of the gene. Three PCR reactions described allowed the analysis of a total of 30 exons and led, to the identification of three additional deletions involving the following exons: (a) 42 only, (b) 28-42, and (c) 16 only, none of which were detected with the two original multiplex reactions. Therefore, the three modified multiplexes detected 95 of the 96 deletions identified among the 152 patients studied so far by using Southern analysis and cDNA probes. The only deletion that remained undetected with this system involves exons 22-25 and generates the junction fragment described elsewhere. The percentage of deletion mutations among DMS/BMD patients amounts to 63%, which is in agreement with similar estimates from other laboratories. When field-inversion gel electrophoresis is coupled to Southern analysis, the detection rate of deletion and duplication mutations reaches 65%.

  10. DMD transcript imbalance determines dystrophin levels.

    Science.gov (United States)

    Spitali, Pietro; van den Bergen, Janneke C; Verhaart, Ingrid E C; Wokke, Beatrijs; Janson, Anneke A M; van den Eijnde, Rani; den Dunnen, Johan T; Laros, Jeroen F J; Verschuuren, Jan J G M; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2013-12-01

    Duchenne and Becker muscular dystrophies are caused by out-of-frame and in-frame mutations, respectively, in the dystrophin encoding DMD gene. Molecular therapies targeting the precursor-mRNA are in clinical trials and show promising results. These approaches will depend on the stability and expression levels of dystrophin mRNA in skeletal muscles and heart. We report that the DMD gene is more highly expressed in heart than in skeletal muscles, in mice and humans. The transcript mutated in the mdx mouse model shows a 5' to 3' imbalance compared with that of its wild-type counterpart and reading frame restoration via antisense-mediated exon skipping does not correct this event. We also report significant transcript instability in 22 patients with Becker dystrophy, clarifying the fact that transcript imbalance is not caused by premature nonsense mutations. Finally, we demonstrate that transcript stability, rather than transcriptional rate, is an important determinant of dystrophin protein levels in patients with Becker dystrophy. We suggest that the availability of the complete transcript is a key factor to determine protein abundance and thus will influence the outcome of mRNA-targeting therapies.

  11. Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method

    Directory of Open Access Journals (Sweden)

    Jean-Paul Iyombe-Engembe

    2016-01-01

    Full Text Available The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD patients, expression of dystrophin (DYS protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel, a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

  12. Restoring Dystrophin Expression in Duchenne Muscular Dystrophy Muscle: Progress in Exon Skipping and Stop Codon Read Through

    OpenAIRE

    Hoffman, Eric P.; Bronson, Abby; Levin, Arthur A.; Takeda, Shin'ichi; Yokota, Toshifumi; Baudy, Andreas R.; Connor, Edward M.

    2011-01-01

    The identification of the Duchenne muscular dystrophy gene and protein in the late 1980s led to high hopes of rapid translation to molecular therapeutics. These hopes were fueled by early reports of delivering new functional genes to dystrophic muscle in mouse models using gene therapy and stem cell transplantation. However, significant barriers have thwarted translation of these approaches to true therapies, including insufficient therapeutic material (eg, cells and viral vectors), challenge...

  13. Parallel synthesis of cell-penetrating peptide conjugates of PMO toward exon skipping enhancement in Duchenne muscular dystrophy.

    Science.gov (United States)

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A; Williams, Donna L; Deuss, Peter; Gait, Michael J

    2015-02-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized. The synthetic methods involve conjugation in solution phase, followed by rapid purification via biotin-streptavidin immobilization and subsequent reductive release into solution, avoiding the need for painstaking high-performance liquid chromatography purifications. The synthesis methods were applied for screening of PMO conjugates of a 16-member library of variants of a 10-residue ApoE peptide, which was suggested for blood-brain barrier crossing. In this work the conjugate library was tested in an exon skipping assay using skeletal mouse mdx cells, a model of Duchene's muscular dystrophy where higher activity peptide-PMO conjugates were identified compared with the starting peptide-PMO. The results demonstrate the power of the parallel synthesis methods for increasing the speed of optimization of peptide sequences in conjugates of PMO for therapeutic screening.

  14. Early-progressive dilated cardiomyopathy in a family with Becker muscular dystrophy related to a novel frameshift mutation in the dystrophin gene exon 27.

    Science.gov (United States)

    Tsuda, Takeshi; Fitzgerald, Kristi; Scavena, Mena; Gidding, Samuel; Cox, Mary O; Marks, Harold; Flanigan, Kevin M; Moore, Steven A

    2015-03-01

    We report a family in which two male siblings with Becker muscular dystrophy (BMD) developed severe dilated cardiomyopathy (DCM) and progressive heart failure (HF) at age 11 years; one died at age 14 years while awaiting heart transplant and the other underwent left ventricular assist device implantation at the same age. Genetic analysis of one sibling showed a novel frameshift mutation in exon 27 of Duchenne muscular dystrophy (DMD) gene (c.3779_3785delCTTTGGAinsGG), in which seven base pairs are deleted and two are inserted. Although this predicts an amino-acid substitution and premature termination (p.Thr1260Argfs*8), muscle biopsy dystrophin immunostaining instead indicates that the mutation is more likely to alter splicing. Despite relatively preserved skeletal muscular performance, both the siblings developed progressive HF secondary to early-onset DCM. In addition, their 7-year-old nephew with delayed gross motor development, mild proximal muscle weakness and markedly elevated serum creatine kinase level (>13 000 IU l(-1)) at 16 months was recently demonstrated to have the familial DMD mutation. Here, we report a novel genotype of BMD with early-onset DCM and progressive lethal HF during early adolescence.

  15. Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment.

    Science.gov (United States)

    Betts, Corinne; Saleh, Amer F; Arzumanov, Andrey A; Hammond, Suzan M; Godfrey, Caroline; Coursindel, Thibault; Gait, Michael J; Wood, Matthew Ja

    2012-08-14

    Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.

  16. Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment

    Directory of Open Access Journals (Sweden)

    Corinne Betts

    2012-01-01

    Full Text Available Antisense oligonucleotides (AOs are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD. AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO: Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.

  17. Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues [v2; ref status: indexed, http://f1000r.es/2dl

    Directory of Open Access Journals (Sweden)

    Liliana Florea

    2013-11-01

    Full Text Available Alternative splicing is widely recognized for its roles in regulating genes and creating gene diversity. However, despite many efforts, the repertoire of gene splicing variation is still incompletely characterized, even in humans. Here we describe a new computational system, ASprofile, and its application to RNA-seq data from Illumina’s Human Body Map project (>2.5 billion reads.  Using the system, we identified putative alternative splicing events in 16 different human tissues, which provide a dynamic picture of splicing variation across the tissues. We detected 26,989 potential exon skipping events representing differences in splicing patterns among the tissues. A large proportion of the events (>60% were novel, involving new exons (~3000, new introns (~16000, or both. When tracing these events across the sixteen tissues, only a small number (4-7% appeared to be differentially expressed (‘switched’ between two tissues, while 30-45% showed little variation, and the remaining 50-65% were not present in one or both tissues compared.  Novel exon skipping events appeared to be slightly less variable than known events, but were more tissue-specific. Our study represents the first effort to build a comprehensive catalog of alternative splicing in normal human tissues from RNA-seq data, while providing insights into the role of alternative splicing in shaping tissue transcriptome differences. The catalog of events and the ASprofile software are freely available from the Zenodo repository (http://zenodo.org/record/7068; doi:10.5281/zenodo.7068 and from our web site http://ccb.jhu.edu/software/ASprofile.

  18. Becker muscular dystrophy due to an intronic splicing mutation inducing a dual dystrophin transcript.

    Science.gov (United States)

    Todeschini, Alice; Gualandi, Francesca; Trabanelli, Cecilia; Armaroli, Annarita; Ravani, Anna; Fanin, Marina; Rota, Silvia; Bello, Luca; Ferlini, Alessandra; Pegoraro, Elena; Padovani, Alessandro; Filosto, Massimiliano

    2016-10-01

    We describe a 29-year-old patient who complained of left thigh muscle weakness since he was 23 and of moderate proximal weakness of both lower limbs with difficulty in climbing stairs and running since he was 27. Mild weakness of iliopsoas and quadriceps muscles and muscle atrophy of both the distal forearm and thigh were observed upon clinical examination. He harboured a novel c.1150-3C>G substitution in the DMD gene, affecting the intron 10 acceptor splice site and causing exon 11 skipping and an out-of-frame transcript. However, protein of normal molecular weight but in reduced amounts was observed on Western Blot analysis. Reverse transcription analysis on muscle RNA showed production, via alternative splicing, of a transcript missing exon 11 as well as a low abundant full-length transcript which is enough to avoid the severe Duchenne phenotype. Our study showed that a reduced amount of full length dystrophin leads to a mild form of Becker muscular dystrophy. These results confirm earlier findings that low amounts of dystrophin can be associated with a milder phenotype, which is promising for therapies aiming at dystrophin restoration. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Gene therapies that restore dystrophin expression for the treatment of Duchenne muscular dystrophy.

    Science.gov (United States)

    Robinson-Hamm, Jacqueline N; Gersbach, Charles A

    2016-09-01

    Duchenne muscular dystrophy is one of the most common inherited genetic diseases and is caused by mutations to the DMD gene that encodes the dystrophin protein. Recent advances in genome editing and gene therapy offer hope for the development of potential therapeutics. Truncated versions of the DMD gene can be delivered to the affected tissues with viral vectors and show promising results in a variety of animal models. Genome editing with the CRISPR/Cas9 system has recently been used to restore dystrophin expression by deleting one or more exons of the DMD gene in patient cells and in a mouse model that led to functional improvement of muscle strength. Exon skipping with oligonucleotides has been successful in several animal models and evaluated in multiple clinical trials. Next-generation oligonucleotide formulations offer significant promise to build on these results. All these approaches to restoring dystrophin expression are encouraging, but many hurdles remain. This review summarizes the current state of these technologies and summarizes considerations for their future development.

  20. PNA-mediated modulation and redirection of Her-2 pre-mRNA splicing: specific skipping of erbB-2 exon 19 coding for the ATP catalytic domain

    DEFF Research Database (Denmark)

    Pankratova, Stanislava; Nielsen, Birgit N; Shiraishi, Takehiko;

    2010-01-01

    The Her-2 receptor coded for by the proto-oncogenic erbB-2 gene is a clinically validated target for treatment of a significant genetic subclass of breast cancers, and Her-2 is also overexpressed or mutated in a range of other cancers. In an approach to exploit antisense mediated splicing...... oligomers that specifically induce skipping of exon 19 as this exon is coding for the ATP catalytic domain of Her-2, and if expressed such truncated version of the Her-2 protein should be functionally inactive in a dominant negative fashion. Therefore, antisense compounds having efficient erbB-2 exon 19...

  1. Skipping of exons by premature termination of transcription and alternative splicing within intron-5 of the sheep SCF gene: a novel splice variant.

    Directory of Open Access Journals (Sweden)

    Siva Arumugam Saravanaperumal

    Full Text Available Stem cell factor (SCF is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM, SCF is produced either as a membrane-bound (- or soluble (+ forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR. Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp cDNA encodes a precursor protein of 274 amino acids (aa, commonly known as 'soluble' isoform. In contrast, the shorter (835 and/or 725 bp cDNA was found to be a 'novel' mRNA splice variant. It contains an open reading frame (ORF corresponding to a truncated protein of 181 aa (vs 245 aa with an unique C-terminus lacking the primary proteolytic segment (28 aa right after the D(175G site which is necessary to produce 'soluble' form of SCF. This alternative splice (AS variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5-exon 6 (948 bp with a premature termination codon (PTC whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6-9/10. We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+ and/or absence (- of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

  2. A one base pair deletion in the canine ATP13A2 gene causes exon skipping and late-onset neuronal ceroid lipofuscinosis in the Tibetan terrier.

    Directory of Open Access Journals (Sweden)

    Anne Wöhlke

    2011-10-01

    Full Text Available Neuronal ceroid lipofuscinosis (NCL is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5-7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA 2 at 83.71-84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9. Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies.

  3. The first exon duplication mouse model of Duchenne muscular dystrophy: A tool for therapeutic development.

    Science.gov (United States)

    Vulin, Adeline; Wein, Nicolas; Simmons, Tabatha R; Rutherford, Andrea M; Findlay, Andrew R; Yurkoski, Jacqueline A; Kaminoh, Yuuki; Flanigan, Kevin M

    2015-11-01

    Exon duplication mutations account for up to 11% of all cases of Duchenne muscular dystrophy (DMD), and a duplication of exon 2 is the most common duplication in patients. For use as a platform for testing of duplication-specific therapies, we developed a mouse model that carries a Dmd exon 2 duplication. By using homologous recombination we duplicated exon 2 within intron 2 at a location consistent with a human duplication hotspot. mRNA analysis confirms the inclusion of a duplicated exon 2 in mouse muscle. Dystrophin expression is essentially absent by immunofluorescent and immunoblot analysis, although some muscle specimens show very low-level trace dystrophin expression. Phenotypically, the mouse shows similarities to mdx, the standard laboratory model of DMD. In skeletal muscle, areas of necrosis and phagocytosis are seen at 3 weeks, with central nucleation prominent by four weeks, recapitulating the "crisis" period in mdx. Marked diaphragm fibrosis is noted by 6 months, and remains unchanged at 12 months. Our results show that the Dup2 mouse is both pathologically (in degree and distribution) and physiologically similar to mdx. As it recapitulates the most common single exon duplication found in DMD patients, this new model will be a useful tool to assess the potential of duplicated exon skipping.

  4. Modeling the human MTM1 p.R69C mutation in murine Mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype

    Science.gov (United States)

    Pierson, Christopher R.; Dulin-Smith, Ashley N.; Durban, Ashley N.; Marshall, Morgan L.; Marshall, Jordan T.; Snyder, Andrew D.; Naiyer, Nada; Gladman, Jordan T.; Chandler, Dawn S.; Lawlor, Michael W.; Buj-Bello, Anna; Dowling, James J.; Beggs, Alan H.

    2012-01-01

    X-linked myotubular myopathy (MTM) is a severe neuromuscular disease of infancy caused by mutations of MTM1, which encodes the phosphoinositide lipid phosphatase, myotubularin. The Mtm1 knockout (KO) mouse has a severe phenotype and its short lifespan (8 weeks) makes it a challenge to use as a model in the testing of certain preclinical therapeutics. Many MTM patients succumb early in life, but some have a more favorable prognosis. We used human genotype–phenotype correlation data to develop a myotubularin-deficient mouse model with a less severe phenotype than is seen in Mtm1 KO mice. We modeled the human c.205C>T point mutation in Mtm1 exon 4, which is predicted to introduce the p.R69C missense change in myotubularin. Hemizygous male Mtm1 p.R69C mice develop early muscle atrophy prior to the onset of weakness at 2 months. The median survival period is 66 weeks. Histopathology shows small myofibers with centrally placed nuclei. Myotubularin protein is undetectably low because the introduced c.205C>T base change induced exon 4 skipping in most mRNAs, leading to premature termination of myotubularin translation. Some full-length Mtm1 mRNA bearing the mutation is present, which provides enough myotubularin activity to account for the relatively mild phenotype, as Mtm1 KO and Mtm1 p.R69C mice have similar muscle phosphatidylinositol 3-phosphate levels. These data explain the basis for phenotypic variability among human patients with MTM1 p.R69C mutations and establish the Mtm1 p.R69C mouse as a valuable model for the disease, as its less severe phenotype will expand the scope of testable preclinical therapies. PMID:22068590

  5. An Intronic SINE insertion in FAM161A that causes exon-skipping is associated with progressive retinal atrophy in Tibetan Spaniels and Tibetan Terriers.

    Directory of Open Access Journals (Sweden)

    Louise M Downs

    Full Text Available Progressive retinal atrophy (PRA in dogs is characterised by the degeneration of the photoreceptor cells of the retina, resulting in vision loss and eventually complete blindness. The condition affects more than 100 dog breeds and is known to be genetically heterogeneous between breeds. Around 19 mutations have now been identified that are associated with PRA in around 49 breeds, but for the majority of breeds the mutation(s responsible have yet to be identified. Using genome-wide association with 22 Tibetan Spaniel PRA cases and 10 controls, we identified a novel PRA locus, PRA3, on CFA10 (p(raw = 2.01 × 10(-5, p(genome = 0.014, where a 3.8 Mb region was homozygous within 12 cases. Using targeted next generation sequencing, a short interspersed nuclear element insertion was identified near a splice acceptor site in an intron of a provocative gene, FAM161A. Analysis of mRNA from an affected dog revealed that the SINE causes exon skipping, resulting in a frame shift, leading to a downstream premature termination codon and possibly a truncated protein product. This mutation segregates with the disease in 22 out of 35 cases tested (63%. Of the PRA controls, none are homozygous for the mutation, 15% carry the mutation and 85% are homozygous wildtype. This mutation was also identified in Tibetan Terriers, although our results indicate that PRA is genetically heterogeneous in both Tibetan Spaniels and Tibetan Terriers.

  6. Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping

    DEFF Research Database (Denmark)

    Madsen, Pia Pinholt; Kibaek, Maria; Roca, Xavier

    2006-01-01

    -causing in both families. Using a minigene approach, we show that the IVS3+3A>G mutation causes exon 3 skipping, despite the fact that it does not appear to disrupt the consensus sequence of the 5' splice site. Based on these results and numerous literature examples, we suggest that this type of mutation (IVS+3A...... of C5-carnitine in blood may indicate SBCADD, the disorder may be detected by MS/MS-based routine newborn screening. It is, therefore, important to gain more knowledge about the clinical presentation and the mutational spectrum of SBCADD. In the present study, we have studied two unrelated families....../MS currently lacks sensitivity in detecting SBCADD. Until now, seven mutations in the SBCAD gene have been reported, but only three have been tested experimentally. Here, we identify and characterize an IVS3+3A>G mutation (c.303+3A>G) in the SBCAD gene, and provide evidence that this mutation is disease...

  7. Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9

    Directory of Open Access Journals (Sweden)

    Hongmei Lisa Li

    2015-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.

  8. Exon-skipping strategy by ratio modulation between cytoprotective versus pro-apoptotic clusterin forms increased sensitivity of LNCaP to cell death.

    Directory of Open Access Journals (Sweden)

    Abdellatif Essabbani

    Full Text Available BACKGROUND: In prostate cancer the secreted form of clusterin (sCLU has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties. METHODOLOGY: In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress. RESULTS AND CONCLUSIONS: We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line "LNCaP" after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an "on demand alternative splicing" strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

  9. Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle

    DEFF Research Database (Denmark)

    Hjortkjær, Camilla Brolin; Shiraishi, Takehiko; Hojman, Pernille;

    2015-01-01

    and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice...... switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find...... that electroporation can enhance PNA antisense effects in muscle tissue....

  10. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin

    Energy Technology Data Exchange (ETDEWEB)

    Thanh, L.T.; Man, Nguyen Thi; Morris, G.E. [Wales Institute, Clwyd (United Kingdom)] [and others

    1995-08-28

    We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immunohistochemistry or Western blotting with these {open_quotes}exon-specific{close_quotes} mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying {open_quotes}revertant{close_quotes} fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene. 27 refs., 7 figs., 3 tabs.

  11. Exon Deletion Pattern in Duchene Muscular Dystrophy in North West of Iran

    Directory of Open Access Journals (Sweden)

    Mohammad BARZEGAR

    2015-01-01

    correlations of deletions leading to Duchenne and Becker muscular dystrophies. Neurology 1989; 39(4:465-474.Koenig M1, Beggs AH, Moyer M, Scherpf S, Heindrich K, Bettecken T, Meng G, Müller CR, Lindlöf M, Kaariainen H, et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 1989; 45(4:498-506.Helderman-van den E, Straathof CSM, Aartsma A, Dendunnen JT, Verbist BM, Bakker E, Verschuuren JJJM, Ginjaar HB. Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients. Neuromuscular disorders 2010; 20:251-254.   Artsma-Rus A, Fokkema I, Verschuuren J, Ginjaar L, Deutekom GV, Ommen GJV et al. Theoretic applicability of antisense-mediated exon skipping for duchenne muscular dystrophy mutations. hummutat 2009; 30:293-9.Van Deutekom JC, Janson AA, Ginjaar LB, Frankhuizen WS, Artsma-Rus A, Bremmer-Bout Mattie et al. Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl Med 2007; 357:2677-86.

  12. HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

    Science.gov (United States)

    Nishida, Atsushi; Yasuno, Sato; Takeuchi, Atsuko; Awano, Hiroyuki; Lee, Tomoko; Niba, Emma Tabe Eko; Fujimoto, Takahiro; Itoh, Kyoko; Takeshima, Yasuhiro; Nishio, Hisahide; Matsuo, Masafumi

    2016-09-01

    The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5' part of the dystrophin cDNA. In contrast, fragments covering exons 64-79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

  13. Analysis of dystrophin gene deletions by multiplex PCR in eastern India

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    Basak Jayasri

    2006-01-01

    Full Text Available The most common genetic neuromuscular disease of childhood, Duchenne and Becker muscular dystrophy (DMD/BMD is caused by deletion, duplication or point mutation of the dystrophin gene located at Xp 21.2. In the present study DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR to screen for exon deletions and its distribution within the dystrophin gene. Out of seventy patients forty six (63% showed large intragenic deletion in the dystrophin gene. About 79% of these deletions are located in the hot spot region i.e., between exon 42 to 53. This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population.

  14. Dystrophin in frameshift deletion patients with Becker Muscular Dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Gangopadhyay, S.B.; Ray, P.N.; Worton, R.G.; Sherratt, T.G.; Heckmatt, J.Z.; Dubowitz, V.; Strong, P.N.; Miller, G. (Penn State College of Medicine, Hershey, PA (United States)); Shokeir, M. (Univ. Hospital, Saskatchewan (Canada))

    1992-09-01

    In a previous study the authors identified 14 cases with Duchenne muscular dystrophy (DMD) or its milder variant, Becker muscular dystrophy (BMD), with a deletion of exons 3-7, a deletion that would be expected to shift the translational reading frame of the mRNA and give a severe phenotype. They have examined dystrophin and its mRNA from muscle biopsies of seven cases with either mild or intermediate phenotypes. In all cases they detected slightly lower-molecular-weight dystrophin in 12%-15% abundance relative to the normal. By sequencing amplified mRNA they have found that exon 2 is spliced to exon 8, a splice that produces a frameshifted mRNA, and have found no evidence for alternate splicing that might be involved in restoration of dystrophin mRNA reading frame in the patients with a mild phenotype. Other transcriptional and posttranscriptional mechanisms such as cryptic promoter, ribosomal frameshifting, and reinitiation are suggested that might play some role in restoring the reading frame. 34 refs., 5 figs. 1 tab.

  15. Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in.

    Science.gov (United States)

    Popplewell, Linda; Koo, Taeyoung; Leclerc, Xavier; Duclert, Aymeric; Mamchaoui, Kamel; Gouble, Agnés; Mouly, Vincent; Voit, Thomas; Pâques, Frédéric; Cédrone, Frédéric; Isman, Olga; Yáñez-Muñoz, Rafael J; Dickson, George

    2013-07-01

    Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.

  16. A Japanese boy with myalgia and cramps has a novel in-frame deletion of the dystrophin gene.

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    Ishigaki, C; Patria, S Y; Nishio, H; Yabe, M; Matsuo, M

    1996-05-01

    We report a Japanese Becker muscular dystrophy (BMD) patient with occasional myalgia and cramps during normal activity that developed at the age of 28 months. His family history was negative for neuromuscular diseases. Muscle biopsy analyses, including dystrophin immunostaining, disclosed no clinically relevant findings. The diagnosis of BMD was initially made at the age of 10 years, when indications of persistent high serum levels of CK prompted us to screen deletions in the dystrophin gene by amplification of 19 deletion-prone exons from the genomic DNA by the polymerase chain reaction (PCR). Among the exons examined, exons 13 and 17 were deleted. To clarify the size of the deletion, the dystrophin transcript was analyzed by reverse transcription PCR. The determined nucleotide sequence of the amplified product encompassing exons 10 to 20 disclosed that the entire segment corresponding to exons 13 to 18 (810 bp) was absent, a deletion that would be expected to cause the production of a dystrophin protein lacking 270 amino acids from the rod domain. This result indicates that occasional myalgia and cramps could be early clinical manifestations of mild BMD, especially in patients who have a deletion in the rod domain, and that deletion screening of the dystrophin gene might be the only reliable method to diagnose such cases.

  17. Spectrum of small mutations in the dystrophin coding region.

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    Prior, T W; Bartolo, C; Pearl, D K; Papp, A C; Snyder, P J; Sedra, M S; Burghes, A H; Mendell, J R

    1995-07-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.

  18. Analysis of Dystrophin Gene Deletions by Multiplex PCR in Moroccan Patients

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    Aziza Sbiti

    2002-01-01

    Full Text Available Duchenne and Becker muscular dystrophy (DMD and BMD are X-linked diseases resulting from a defect in the dystrophin gene located on Xp21. DMD is the most frequent neuromuscular disease in humans (1/3500 male newborn. Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. We have analyzed DNA from 72 Moroccan patients with DMD/BMD using the multiplex polymerase chain reaction (PCR to screen for exon deletions within the dystrophin gene, and to estimate the frequency of these abnormalities. We found dystrophin gene deletions in 37 cases. Therefore the frequency in Moroccan DMD/BMD patients is about 51.3%. All deletions were clustered in the two known hot-spots regions, and in 81% of cases deletions were detected in the region from exon 43 to exon 52. These findings are comparable to those reported in other studies. It is important to note that in our population, we can first search for deletions of DMD gene in the most frequently deleted exons determined by this study. This may facilitate the molecular diagnosis of DMD and BMD in our country.

  19. Is the human dystrophin gene's intron structure related to its intron instability?

    Institute of Scientific and Technical Information of China (English)

    盛文利; 陈江瑛; 朱良付; 刘焯霖

    2003-01-01

    Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34.8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.

  20. Dystrophin-Deficient Cardiomyopathy.

    Science.gov (United States)

    Kamdar, Forum; Garry, Daniel J

    2016-05-31

    Dystrophinopathies are a group of distinct neuromuscular diseases that result from mutations in the structural cytoskeletal Dystrophin gene. Dystrophinopathies include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, as well as DMD and BMD female carriers. The primary presenting symptom in most dystrophinopathies is skeletal muscle weakness. However, cardiac muscle is also a subtype of striated muscle and is similarly affected in many of the muscular dystrophies. Cardiomyopathies associated with dystrophinopathies are an increasingly recognized manifestation of these neuromuscular disorders and contribute significantly to their morbidity and mortality. Recent studies suggest that these patient populations would benefit from cardiovascular therapies, annual cardiovascular imaging studies, and close follow-up with cardiovascular specialists. Moreover, patients with DMD and BMD who develop end-stage heart failure may benefit from the use of advanced therapies. This review focuses on the pathophysiology, cardiac involvement, and treatment of cardiomyopathy in the dystrophic patient. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  1. Relatively low proportion of dystrophin gene deletions in Israeili Duchenne and Becker muscular dystrophy patients

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    Shomrat, R.; Gluck, E.; Legum, C.; Shiloh, Y. [Tel Aviv Univ. (Israel)

    1994-02-15

    Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the X-linked dystrophin gene. The most common mutations in western populations are deletions that are spread non-randomly throughout the gene. Molecular analysis of the dystrophin gene structure by hybridization of the full length cDNA to Southern blots and by PCR in 62 unrelated Israeli male DMD/BMD patients showed deletions in 23 (37%). This proportion is significantly lower than that found in European and North American populations (55-65%). Seventy-eight percent of the deletions were confined to exons 44-52, half of these exons 44-45, and the remaining 22% to exons 1 and 19. There was no correlation between the size of the deletion and the severity of the disease. All the deletions causing frameshift resulted in the DMD phenotypes. 43 refs., 1 fig., 1 tab.

  2. Molecular Diagnosis of Duchenne/Becker Muscular Dystrophy: Analysis of Exons Deletion and Carrier Detection

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    Mohammad Taghi Akbari

    2010-01-01

    Full Text Available Objective: Duchenne and Becker Muscular Dystrophy (DMD and BMD are X-linked conditionsresulting from a defect in the dystrophin gene located at Xp21.2. DMD is the mostfrequent neuromuscular disease in humans (1/3500 male newborns. In approximately65% of DMD and BMD patients, deletions in the dystrophin gene have been identified asthe molecular determinant. The frequency and distribution of dystrophin gene deletions inDMD/BMD patients from different populations are different.The aim of this study was to delineate various types of deleted exons and their frequencyin affected male patients and identification of carrier females by linkage analysis.Materials and Methods: In this study 100 unrelated patients with DMD/BMD were studiedfor intragenic deletions in 28 exons and the promoter region of the dystrophin geneusing multiplex PCR. We also performed linkage analysis within the dystrophin gene utilizing8 short tandem repeat markers.Results: Fifty-two (52% patients showed intragenic deletions. A total of 81% of the deletionswere located at the distal hot spot region (44-55 exons and 19% of the deletionswere located at the proximal region (exon 2-19. The most frequent deleted exons were47(16%, 48 and 46 (11%.Most of the STR markers showed heterozygosity in the families studied. The linkageanalysis was useful for detecting carrier status.Conclusion: The present study suggests that intragenic dystrophin gene deletions occurwith the same frequency in Iranian patients compared with other ethnic groups.

  3. Gaucher disease: A G[sup +1][yields]A[sup +1] IVS2 splice donor site mutation causing exon 2 skipping in the acid [beta]-glucosidase mRNA

    Energy Technology Data Exchange (ETDEWEB)

    He, Guo-Shun (Mount Siani School of Medicine, New York, NY (United States)); Grabowski, G.A. (Children' s Hospital Medical Center, Cincinnati, OH (United States))

    1992-10-01

    Gaucher disease is the most frequent lysosomal storage disease and the most prevalent Jewish genetic disease. About 30 identified missense mutations are causal to the defective activity of acid [beta]-glucosidase in this disease. cDNAs were characterized from a moderately affected 9-year-old Ashkenazi Jewish Gaucher disease type 1 patient whose 80-years-old, enzyme-deficient, 1226G (Asn[sup 370][yields]Ser [N370S]) homozygous grandfather was nearly asymptomatic. Sequence analyses revealed four populations of cDNAs with either the 1226G mutation, an exact exon 2 ([Delta] EX2) deletion, a deletion of exon 2 and the first 115 bp of exon 3 ([Delta] EX2-3), or a completely normal sequence. About 50% of the cDNAs were the [Delta] EX2, the [Delta] EX2-3, and the normal cDNAs, in a ratio of 6:3:1. Specific amplification and characterization of exon 2 and 5[prime] and 3[prime] intronic flanking sequences from the structural gene demonstrated clones with either the normal sequence or with a G[sup +1][yields]A[sup +1] transition at the exon 2/intron 2 boundary. This mutation destroyed the splice donor consensus site (U1 binding site) for mRNA processing. This transition also was present at the corresponding exon/intron boundary of the highly homologous pseudogene. This new mutation, termed [open quotes]IVS2 G[sup +1],[close quotes] is the first in the Ashkenazi Jewish population. The occurrence of this [open quotes]pseudogene[close quotes]-type mutation in the structural gene indicates the role of acid [beta]-glucosidase pseudogene and structural gene rearrangements in the pathogenesis of this disease. 33 refs., 8 figs., 1 tab.

  4. Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database

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    Nicolas Aurélie

    2012-07-01

    Full Text Available Abstract Background Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD and Becker (BMD muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. Methods and results We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. Conclusion This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.

  5. Heteroduplex analysis of the dystrophin gene: application to point mutation and carrier detection.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Sedra, M S; Western, L M; Bartolo, C; Moxley, R T; Mendell, J R

    1994-03-01

    Approximately one-third of the Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, we identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. We conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing.

  6. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    Energy Technology Data Exchange (ETDEWEB)

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R. [Ohio State Univ., Columbus, OH (United States); Moxley, R.T. [Univ. of Rochester Medical Center, NY (United States)

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  7. Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot.

    Science.gov (United States)

    Kodippili, Kasun; Vince, Lauren; Shin, Jin-Hong; Yue, Yongping; Morris, Glenn E; McIntosh, Mark A; Duan, Dongsheng

    2014-01-01

    Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model.

  8. Becker muscular dystrophy in Indian patients: Analysis of dystrophin gene deletion patterns

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    Dastur Rashna

    2008-01-01

    Full Text Available Background: Becker muscular dystrophy (BMD is caused by mutations in the dystrophin gene with variable phenotypes. Becker muscular dystrophy patients have low levels of nearly full-length dystrophin and carry in-frame mutations, which allow partial functioning of the protein. Aim: To study the deletion patterns of BMD and to correlate the same with reading frame rule and different phenotypes. Setting: A tertiary care teaching hospital. Design: This is a prospective hospital-based study. Materials and Methods: Thirty-two exons spanning different "hot spot" regions using Multiplex PCR techniques were studied in 347 patients. Two hundred and twenty-two showed deletions in one or more of the 32 exons. Out of these, 46 diagnosed as BMD patients were analyzed. Results: Forty-six BMD patients showed deletions in both regions of the dystrophin gene. Out of these 89.1% (41/46 were in-frame deletions. Deletions starting with Exon 45 were found in 76.1% (35/46 of the cases. Mutations in the majority of cases i.e. 39/46 (84.8% were seen in 3′ downstream region (Exon 45-55, distal rod domain. Few, i.e. 5/46 (10.8% showed deletions in 5′ upstream region (Exons 3-20, N-terminus and proximal rod domain of the gene, while in 2/46 (4.4% large mutations (>40 bp spanning both regions (Exons 3-55 were detected. Conclusion: This significant gene deletion analysis has been carried out for BMD patients particularly from Western India using 32 exons.

  9. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

    Science.gov (United States)

    Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja

    2010-10-01

    Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

  10. Stability in skipping gaits

    Science.gov (United States)

    Andrada, Emanuel; Müller, Roy; Blickhan, Reinhard

    2016-11-01

    As an alternative to walking and running, humans are able to skip. However, adult humans avoid it. This fact seems to be related to the higher energetic costs associated with skipping. Still, children, some birds, lemurs and lizards use skipping gaits during daily locomotion. We combined experimental data on humans with numerical simulations to test whether stability and robustness motivate this choice. Parameters for modelling were obtained from 10 male subjects. They locomoted using unilateral skipping along a 12 m runway. We used a bipedal spring loaded inverted pendulum to model and to describe the dynamics of skipping. The subjects displayed higher peak ground reaction forces and leg stiffness in the first landing leg (trailing leg) compared to the second landing leg (leading leg). In numerical simulations, we found that skipping is stable across an amazing speed range from skipping on the spot to fast running speeds. Higher leg stiffness in the trailing leg permits longer strides at same system energy. However, this strategy is at the same time less robust to sudden drop perturbations than skipping with a stiffer leading leg. A slightly higher stiffness in the leading leg is most robust, but might be costlier.

  11. Screening of Dystrophin Gene Deletions in Egyptian Patients with DMD/BMD Muscular Dystrophies

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    Laila K. Effat

    2000-01-01

    Full Text Available Duchenne muscular dystrophy (DMD and Becker muscular dystrophy (BMD are allelic disorders caused by mutations within the dystrophin gene. Our study has identified 100 Egyptian families collected from the Human Genetics Clinic, National Research Center, Cairo. All cases were subjected to complete clinical evaluation pedigree analysis, electromyography studies, estimation of serum creatine phosphokinase enzyme (CPK levels and DNA analysis. Multiplex PCR using 18 pairs of specific primers were used for screening of deletion mutations within the dystrophin gene. A frequency of 55% among the families. Sixty per cent of detected deletions involved multiple exons spanning the major or the minor hot spot of the dystrophin gene. The remainder 40% which mainly involved exon 45. Comparing these findings with frequencies of other countries it was found that our figures fall within the reported range of 40%– for deletions. The distribution of deletions in our study and other different studies was variable and specific ethnic differences do not apparently account for specific deletions. In addition this study concluded that employment of the 18 exon analysis is a cost effective and a highly accurate (97% to launch a nationwide program.

  12. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  13. Dystrophin and the two related genetic diseases, Duchenne and Becker muscular dystrophies

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    Elisabeth Le Rumeur

    2015-07-01

    Full Text Available Mutations of the dystrophin DMD gene, essentially deletions of one or several exons, are the cause of two devastating and to date incurable diseases, Duchenne (DMD and Becker (BMD muscular dystrophies. Depending upon the preservation or not of the reading frame, dystrophin is completely absent in DMD, or present in either a mutated or a truncated form in BMD. DMD is a severe disease which leads to a premature death of the patients. Therapy approaches are evolving with the aim to transform the severe DMD in the BMD form of the disease by restoring the expression of a mutated or truncated dystrophin. These therapies are based on the assumption that BMD is a mild disease. However, this is not completely true as BMD patients are more or less severely affected and no molecular basis of this heterogeneity of the BMD form of the disease is yet understood. The aim of this review is to report for the correlation between dystrophin structures in BMD deletions in view of this heterogeneity and to emphasize that examining BMD patients in details is highly relevant to anticipate for DMD therapy effects.

  14. Predicting mutually exclusive spliced exons based on exon length, splice site and reading frame conservation, and exon sequence homology

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    Hammesfahr Björn

    2011-06-01

    Full Text Available Abstract Background Alternative splicing of pre-mature RNA is an important process eukaryotes utilize to increase their repertoire of different protein products. Several types of different alternative splice forms exist including exon skipping, differential splicing of exons at their 3'- or 5'-end, intron retention, and mutually exclusive splicing. The latter term is used for clusters of internal exons that are spliced in a mutually exclusive manner. Results We have implemented an extension to the WebScipio software to search for mutually exclusive exons. Here, the search is based on the precondition that mutually exclusive exons encode regions of the same structural part of the protein product. This precondition provides restrictions to the search for candidate exons concerning their length, splice site conservation and reading frame preservation, and overall homology. Mutually exclusive exons that are not homologous and not of about the same length will not be found. Using the new algorithm, mutually exclusive exons in several example genes, a dynein heavy chain, a muscle myosin heavy chain, and Dscam were correctly identified. In addition, the algorithm was applied to the whole Drosophila melanogaster X chromosome and the results were compared to the Flybase annotation and an ab initio prediction. Clusters of mutually exclusive exons might be subsequent to each other and might encode dozens of exons. Conclusions This is the first implementation of an automatic search for mutually exclusive exons in eukaryotes. Exons are predicted and reconstructed in the same run providing the complete gene structure for the protein query of interest. WebScipio offers high quality gene structure figures with the clusters of mutually exclusive exons colour-coded, and several analysis tools for further manual inspection. The genome scale analysis of all genes of the Drosophila melanogaster X chromosome showed that WebScipio is able to find all but two of the 28

  15. Intellectual Ability in the Duchenne Muscular Dystrophy and Dystrophin Gene Mutation Location

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    Rasic Milic V.

    2014-12-01

    Full Text Available Duchenne muscular dystrophy (DMD is the most common form of muscular dystrophy during childhood. Mutations in dystrophin (DMD gene are also recognized as a cause of cognitive impairment. We aimed to determine the association between intelligence level and mutation location in DMD genes in Serbian patients with DMD. Forty-one male patients with DMD, aged 3 to 16 years, were recruited at the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia. All patients had defined DMD gene deletions or duplications [multiplex ligation- dependent probe amplification (MLPA, polymerase chain reaction (PCR] and cognitive status assessment (Wechsler Intelligence Scale for Children, Brunet-Lezine scale, Vineland-Doll scale. In 37 patients with an estimated full scale intelligence quotient (FSIQ, six (16.22% had borderline intelligence (70exons 30 and 45. However, FSIQ was statistically significantly associated with mutation location when we assumed their functional consequence on dystrophin isoforms and when mutations in the 5’-untranslated region (5’UTR of Dp140 (exons 45-50 were assigned to affect only Dp427 and Dp260. Mutations affecting Dp140 and Dp71/Dp40 have been associated with more frequent and more severe cognitive impairment. Finally, the same classification of mutations explained the greater proportion of FSIQ variability associated with cumulative loss of dystrophin isoforms. In conclusion, cumulative loss of dystrophin isoforms increases the risk of intellectual impairment in DMD and characterizing the genotype can define necessity of early cognitive interventions in DMD patients.

  16. NextSearch: A Search Engine for Mass Spectrometry Data against a Compact Nucleotide Exon Graph.

    Science.gov (United States)

    Kim, Hyunwoo; Park, Heejin; Paek, Eunok

    2015-07-02

    Proteogenomics research has been using six-frame translation of the whole genome or amino acid exon graphs to overcome the limitations of reference protein sequence database; however, six-frame translation is not suitable for annotating genes that span over multiple exons, and amino acid exon graphs are not convenient to represent novel splice variants and exon skipping events between exons of incompatible reading frames. We propose a proteogenomic pipeline NextSearch (Nucleotide EXon-graph Transcriptome Search) that is based on a nucleotide exon graph. This pipeline consists of constructing a compact nucleotide exon graph that systematically incorporates novel splice variations and a search tool that identifies peptides by directly searching the nucleotide exon graph against tandem mass spectra. Because our exon graph stores nucleotide sequences, it can easily represent novel splice variations and exon skipping events between incompatible reading frame exons. Searching for peptide identification is performed against this nucleotide exon graph, without converting it into a protein sequence in FASTA format, achieving an order of magnitude reduction in the size of the sequence database storage. NextSearch outputs the proteome-genome/transcriptome mapping results in a general feature format (GFF) file, which can be visualized by public tools such as the UCSC Genome Browser.

  17. A defect in dystrophin causes a novel porcine stress syndrome

    Directory of Open Access Journals (Sweden)

    Nonneman Dan J

    2012-06-01

    Full Text Available Abstract Background Losses of slaughter-weight pigs due to transport stress are both welfare and economic concerns to pork producers. Historically, the HAL-1843 mutation in ryanodine receptor 1 was considered responsible for most of the losses; however, DNA testing has effectively eliminated this mutation from commercial herds. We identified two sibling barrows in the USMARC swine herd that died from apparent symptoms of a stress syndrome after transport at 12 weeks of age. The symptoms included open-mouth breathing, skin discoloration, vocalization and loss of mobility. Results We repeated the original mating along with sire-daughter matings to produce additional offspring. At 8 weeks of age, heart rate and electrocardiographs (ECG were monitored during isoflurane anesthesia challenge (3% for 3 min. Four males from the original sire-dam mating and two males from a sire-daughter mating died after one minute of anesthesia. Animals from additional litters were identified as having a stress response, sometimes resulting in death, during regular processing and weighing. Affected animals had elevated plasma creatine phosphokinase (CPK levels before and immediately after isoflurane challenge and cardiac arrhythmias. A pedigree containing 250 pigs, including 49 affected animals, was genotyped with the Illumina PorcineSNP60 Beadchip and only one chromosomal region, SSCX at 25.1-27.7 Mb over the dystrophin gene (DMD, was significantly associated with the syndrome. An arginine to tryptophan (R1958W polymorphism in exon 41 of DMD was the most significant marker associated with stress susceptibility. Immunoblots of affected heart and skeletal muscle showed a dramatic reduction of dystrophin protein and histopathology of affected hearts indicated muscle fiber degeneration. Conclusions A novel stress syndrome was characterized in pigs and the causative genetic factor most likely resides within DMD that results in less dystrophin protein and cardiac

  18. Grade Skipping in Gifted Education

    Institute of Scientific and Technical Information of China (English)

    徐云婕

    2016-01-01

    Grade skipping has been heatedly discussed with the development of gifted education. Experts and scholars are trying to do ifnd out the better way to cultivate those gifted children, to develop their potential, and make full use of their talent. Although grade skipping has long history, there is no certain comment about whether it is beneiftial to the gifted children.

  19. Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2

    DEFF Research Database (Denmark)

    Witting, Nanna; Duno, M; Vissing, J

    2013-01-01

    Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic......, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest...

  20. An RRM-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion.

    Science.gov (United States)

    Collins, Katherine M; Kainov, Yaroslav A; Christodolou, Evangelos; Ray, Debashish; Morris, Quaid; Hughes, Timothy; Taylor, Ian A; Makeyev, Eugene V; Ramos, Andres

    2017-04-04

    RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3΄ intronic sites only. Here, we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1-ZnF unit recognizes a GGA-centered motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognizes a C-rich sequence, which explains its known interaction with the intronic 3΄ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10's broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping.

  1. Stemcell Information: SKIP000253 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000253 ... Diseased GM23760 GM23760 ... 統合失調症 F20 Schizophrenia 181500 ... ...(SCZD) .episodes of agitatation, delusions of persecutation, fear of assassination, father also affected. 統合

  2. Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Richard S Finkel

    Full Text Available BACKGROUND: Approximately 13% of boys with Duchenne muscular dystrophy (DMD have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124 enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. METHODS: This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6 received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20 was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12 was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. FINDINGS: Twenty three of 38 (61% subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. INTERPRETATION: Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. TRIAL REGISTRATION: ClinicalTrials.gov NCT00264888.

  3. Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy.

    Science.gov (United States)

    Finkel, Richard S; Flanigan, Kevin M; Wong, Brenda; Bönnemann, Carsten; Sampson, Jacinda; Sweeney, H Lee; Reha, Allen; Northcutt, Valerie J; Elfring, Gary; Barth, Jay; Peltz, Stuart W

    2013-01-01

    Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124) enables ribosomal readthrough of premature stop codons, leading to production of full-length, functional proteins. This Phase 2a open-label, sequential dose-ranging trial recruited 38 boys with nonsense mutation DMD. The first cohort (n = 6) received ataluren three times per day at morning, midday, and evening doses of 4, 4, and 8 mg/kg; the second cohort (n = 20) was dosed at 10, 10, 20 mg/kg; and the third cohort (n = 12) was dosed at 20, 20, 40 mg/kg. Treatment duration was 28 days. Change in full-length dystrophin expression, as assessed by immunostaining in pre- and post-treatment muscle biopsy specimens, was the primary endpoint. Twenty three of 38 (61%) subjects demonstrated increases in post-treatment dystrophin expression in a quantitative analysis assessing the ratio of dystrophin/spectrin. A qualitative analysis also showed positive changes in dystrophin expression. Expression was not associated with nonsense mutation type or exon location. Ataluren trough plasma concentrations active in the mdx mouse model were consistently achieved at the mid- and high- dose levels in participants. Ataluren was generally well tolerated. Ataluren showed activity and safety in this short-term study, supporting evaluation of ataluren 10, 10, 20 mg/kg and 20, 20, 40 mg/kg in a Phase 2b, double-blind, long-term study in nonsense mutation DMD. ClinicalTrials.gov NCT00264888.

  4. Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

    Science.gov (United States)

    Di Giacomo, Daniela; Gaildrat, Pascaline; Abuli, Anna; Abdat, Julie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2013-11-01

    Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing.

  5. Exon 44 nonsense mutation in two-Duchenne muscular dystrophy brothers detected by heteroduplex analysis.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartolo, C; Mendell, J R

    1993-01-01

    Utilizing a heteroduplex method, we screened the dystrophin exon 43-45 region for point mutations, including small deletions and insertions. The method depends upon the formation of a heteroduplex between wild-type and mutant DNA PCR products. DNA specimens from one hundred and four DMD patients without detected deletions or duplications were multiplexed amplified for exons 43, 44, and 45. The PCR products were mixed with the PCR products from nonaffected controls, electrophoresed, and examined for the presence of altered mobility heteroduplex bands. An exon 44 nonsense mutation in two DMD brothers and a common intron 44 polymorphism were identified using this approach. Although the exon 44-45 region is a hotspot for deletion breakpoints, it does not appear to be prone to point mutations. The technique is extremely useful for screening several exons simultaneously and it allowed us to screen a large number of patients.

  6. Exon silencing by UAGG motifs in response to neuronal excitation.

    Directory of Open Access Journals (Sweden)

    Ping An

    2007-02-01

    Full Text Available Alternative pre-mRNA splicing plays fundamental roles in neurons by generating functional diversity in proteins associated with the communication and connectivity of the synapse. The CI cassette of the NMDA R1 receptor is one of a variety of exons that show an increase in exon skipping in response to cell excitation, but the molecular nature of this splicing responsiveness is not yet understood. Here we investigate the molecular basis for the induced changes in splicing of the CI cassette exon in primary rat cortical cultures in response to KCl-induced depolarization using an expression assay with a tight neuron-specific readout. In this system, exon silencing in response to neuronal excitation was mediated by multiple UAGG-type silencing motifs, and transfer of the motifs to a constitutive exon conferred a similar responsiveness by gain of function. Biochemical analysis of protein binding to UAGG motifs in extracts prepared from treated and mock-treated cortical cultures showed an increase in nuclear hnRNP A1-RNA binding activity in parallel with excitation. Evidence for the role of the NMDA receptor and calcium signaling in the induced splicing response was shown by the use of specific antagonists, as well as cell-permeable inhibitors of signaling pathways. Finally, a wider role for exon-skipping responsiveness is shown to involve additional exons with UAGG-related silencing motifs, and transcripts involved in synaptic functions. These results suggest that, at the post-transcriptional level, excitable exons such as the CI cassette may be involved in strategies by which neurons mount adaptive responses to hyperstimulation.

  7. Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable - Molecular pathology of mutations in PAH exon 11

    DEFF Research Database (Denmark)

    Heintz, Caroline; Dobrowolski, Steven F.; Andersen, Henriette Skovgaard

    2012-01-01

    as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T......-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response. Copyright © 2012 Elsevier Inc. All rights reserved. PMID:22698810[PubMed - in process]...

  8. Laryngeal Muscles Are Spared in the Dystrophin Deficient "mdx" Mouse

    Science.gov (United States)

    Thomas, Lisa B.; Joseph, Gayle L.; Adkins, Tracey D.; Andrade, Francisco H.; Stemple, Joseph C.

    2008-01-01

    Purpose: "Duchenne muscular dystrophy (DMD)" is caused by the loss of the cytoskeletal protein, dystrophin. The disease leads to severe and progressive skeletal muscle wasting. Interestingly, the disease spares some muscles. The purpose of the study was to determine the effects of dystrophin deficiency on 2 intrinsic laryngeal muscles, the…

  9. Sensitivity and Frequencies of Dystrophin Gene Mutations in Thai DMD/BMD Patients As Detected by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    Thanyachai Sura

    2008-01-01

    Full Text Available Background: Duchenne muscular dystrophy (DMD, a lethal X-linked disease affecting 1 in 3500 male births, and its more benign variant, Becker muscular dystrophy (BMD, are caused by mutations in the dystrophin gene. Because of its large size, analysing the whole gene is impractical. Methods have been developed to detect the commonest mutations i.e. the deletions of the exons. Although these tests are highly specific, their sensitivity is inherently limited by the prevalence of deletions, which differs among different populations.

  10. Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle

    Science.gov (United States)

    Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency while Becker muscular dystrophy (BMD) is caused by a dystrophin insufficiency or expression of a partially functional protein product. Both of these dystrophinopathies are most commonly studied using the mdx mouse and a golden r...

  11. TALE-directed local modulation of H3K9 methylation shapes exon recognition.

    Science.gov (United States)

    Bieberstein, Nicole I; Kozáková, Eva; Huranová, Martina; Thakur, Prasoon K; Krchňáková, Zuzana; Krausová, Michaela; Carrillo Oesterreich, Fernando; Staněk, David

    2016-07-21

    In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

  12. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

    Science.gov (United States)

    Bengtsson, Niclas E; Hall, John K; Odom, Guy L; Phelps, Michael P; Andrus, Colin R; Hawkins, R David; Hauschka, Stephen D; Chamberlain, Joel R; Chamberlain, Jeffrey S

    2017-02-14

    Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx(4cv) mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.

  13. Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Thibaut Larcher

    Full Text Available A few animal models of Duchenne muscular dystrophy (DMD are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD.

  14. Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy.

    Science.gov (United States)

    Larcher, Thibaut; Lafoux, Aude; Tesson, Laurent; Remy, Séverine; Thepenier, Virginie; François, Virginie; Le Guiner, Caroline; Goubin, Helicia; Dutilleul, Maéva; Guigand, Lydie; Toumaniantz, Gilles; De Cian, Anne; Boix, Charlotte; Renaud, Jean-Baptiste; Cherel, Yan; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio; Huchet, Corinne

    2014-01-01

    A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmdmdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmdmdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmdmdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmdmdx rats represent a new faithful small animal model of DMD.

  15. Stemcell Information: SKIP000261 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000261 ... Diseased GM23762 GM23762 ... 統合失調症 F20 Schizophrenia 181500 ... ...cally affected with Shizophrenia(SCZD) . 統合失調症患者線維芽細胞(GM02497)由来iPS細胞株。 | human ES-like -- Retrovirus Oct4,

  16. Stemcell Information: SKIP001116 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001116 ... Diseased D2-2 D2-2 ... 統合失調症 F209 schizophrenia ... 605210 ... 39 ...upted in schizophrenia 1 ( DISC1 ) 統合失調症患者由来iPS細胞 human ES-like Research Grade Plasmid OCT4,SOX2,KLF4,c-MYC,

  17. Stemcell Information: SKIP001115 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001115 ... Diseased D2-1 D2-1 ... 統合失調症 F209 schizophrenia ... 605210 ... 39 ...upted in schizophrenia 1 ( DISC1 ) 統合失調症患者由来iPS細胞 human ES-like Research Grade Plasmid OCT4,SOX2,KLF4,c-MYC,

  18. Stemcell Information: SKIP000260 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000260 ... Diseased GM23761 GM23761 ... 統合失調症 F20 Schizophrenia 181500 ... ...h Shizophrenia(SCZD) . | 統合失調症患者線維芽細胞(GM01835)由来iPS細胞株| human ES-like -- Lentivirus Oct4, Sox2, Klf4, c-Myc,

  19. Stemcell Information: SKIP000858 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000858 ... αβT αβT Normal 585A1 585A1 ... 30 30-39 Male Japanese Japanese -- No hiPSC Generat...ion from Peripheral Blood.Medium contained IL-2 and antibodies against CD3 and CD28

  20. Stemcell Information: SKIP000859 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000859 ... αβT αβT Normal 585B1 585B1 ... 30 30-39 Male Japanese Japanese -- No hiPSC Generat...ion from Peripheral Blood.Medium contained IL-2 and antibodies against CD3 and CD28

  1. Stemcell Information: SKIP000845 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000845 ... unknown 不明 Diseased Sporadic PD-1 Sporadic PD-1 10005.117.01 10005.117.01 パーキンソン...oradic Parkinson Disease patient.Using CytoTune iPS Sendai reprogramming protocol (Life Technologies). 孤発性パーキンソン

  2. Stemcell Information: SKIP000265 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000265 ... Diseased HPS0264 HPS0264 ... パーキンソン病 G20 Parkinson disease ... ... ... 40-49 Male ... Yes No iPS cell line derived from Parkinson disease patient. パーキンソン病患者由来| human ES-like -- Re

  3. Stemcell Information: SKIP000279 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000279 ... Diseased HPS0192 HPS0192 ... 杉花粉症 J301 Allergic rhinitis (Pollen allergy) 607154 食物...アレルギー(カニ) T781 food allergy (crab) ... -- -- Japanese Japanese No No Disease specific iPS

  4. Stemcell Information: SKIP000189 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000189 ... Diseased GM23404 GM23404 ... フリードライヒ運動失調症 G111 Friedreich ataxia (a...sitory http://ccr.coriell.org/Sections/Collections/NIGMS/ipsc_list.aspx?PgId=696 ... 21040903 10.1016/j.stem.2010.09.014 Friedrei

  5. Stemcell Information: SKIP000245 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000245 ... Diseased HPS0164 HPS0164 ... デュシェンヌ型筋ジストロフィー G710 Duchenne Muscular... Dystrophy 310200 ... -- -- ... Yes No intractable disease-specific iPSC derived from Duchenne Muscular Dystrophy. デュシェンヌ

  6. Stemcell Information: SKIP000916 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000916 ... fibroblast 線維芽細胞 Normal WT#5J WT#5J ... -- -- ... -- No iPS cell line iPS cell...essful disease-specific induced pluripotent stem cell generation from patients with kidney transplantation.

  7. Stemcell Information: SKIP001066 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001066 ... Diseased LQT3 LQT3 ... QT延長症候群 I45.81 Long QT Syndrome 192500 ... ... ... -- Unknown ... Yes No Specific disease iPS cell line from patient with Long QT Syndrom QT延長症候群患者由来iPS細胞 hu

  8. Stemcell Information: SKIP001117 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001117 ... Diseased D3-1 D3-1 ... 大うつ病 F33.3 major depression 605210 ... ...-- Male ... Yes No iPS cells derived from fibroblasts of a patient in which a frameshift mutation of disrupted in major depression

  9. Stemcell Information: SKIP001118 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001118 ... Diseased D3-2 D3-2 ... 大うつ病 F33.3 major depression 605210 ... ...-- Male ... Yes No iPS cells derived from fibroblasts of a patient in which a frameshift mutation of disrupted in major depression

  10. Stemcell Information: SKIP000269 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ease specific iPS cell line derived from a patient: Mitochondrial myopathy, Encephalopathy, Lactic acidosis,...Encephalopathy, Lactic acidosis, and Stroke-like episodes 540000 ... -- -- Japanese Japanese Yes No Dis... SKIP000269 ... Diseased HPS0070 HPS0070 ... ミトコンドリア病 E888 Mitochondrial myopathy,

  11. Stemcell Information: SKIP000095 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000095 ... Diseased A000052#3 A000052#3 ... 高乳酸血症 R798 hyperlactacidemia ... ... ... -- -- ... Yes No intractable disease-specific iPSC, congenital hyperlactacidemia, suspicous 難病性疾患克服事業iPS細胞バンク 先天性高乳酸血症

  12. Stemcell Information: SKIP000733 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000733 ... Diseased GM24666 GM24666 sAD2 sAD2 アルツハイマー病 G309 ALZHEIMER DISEAS...E 104300 ... 83 80-89 Male ... Yes Yes ALZHEIMER DISEASE(Sporadic AD) hiPSC derived from fibroblast アルツハイマー

  13. Stemcell Information: SKIP000856 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000856 ... Diseased HPS0256 HPS0256 ... アルツハイマー病 G309 Alzheimer disease 104300... ... -- -- ... No No Disease specific iPS cell line derived from a patient with Alzheimer disease アルツハイマー

  14. Stemcell Information: SKIP000855 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000855 ... Diseased HPS0255 HPS0255 ... アルツハイマー病 G309 Alzheimer disease 104300... ... -- -- Japanese Japanese No No Disease specific iPS cell line derived from a patient with Alzheimer disease アルツハイマー

  15. Stemcell Information: SKIP000854 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000854 ... Diseased HPS0254 HPS0254 ... アルツハイマー病 G309 Alzheimer disease 104300... ... -- -- Japanese Japanese No No Disease specific iPS cell line derived from a patient with Alzheimer disease アルツハイマー

  16. Stemcell Information: SKIP000239 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000239 ... Diseased HPS0214 HPS0214 ... エーラース・ダンロス症候群 Q796 Ehlers-Danlos syndrome... 130000 ... -- -- ... Yes No intractable disease-specific iPSC derived from Ehlers-Danlos syndrome, ne

  17. Stemcell Information: SKIP000555 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000555 ... Diseased HPS0191 HPS0191 ... ベルナール・スーリエ症候群 D69.1 Bernard-Soulier syndrome... 231200 ... -- -- ... No No Disease specific iPS cell line derived from Bernard-Soulier syndrome patient ベルナール·スーリエ症候群

  18. Quantitative analysis of the dystrophin gene by real-time PCR

    Directory of Open Access Journals (Sweden)

    Maksimovic Nela

    2012-01-01

    Full Text Available Duchenne and Becker muscular dystrophy (DMD/BMD are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR® Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (ΔΔCt. The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07±0.20, and for carriers 0.56±0.11. This assay proved to be simple, rapid, reliable and cost-effective.

  19. A founder synonymous COL7A1 mutation in three Danish families with dominant dystrophic epidermolysis bullosa pruriginosa identifies exonic regulatory sequences required for exon 87 splicing

    DEFF Research Database (Denmark)

    Covaciu, C; Grosso, F; Pisaneschi, E

    2011-01-01

    a previously unrecognized translationally silent exonic COL7A1 mutation that results in skipping of exon 87 and is associated with DDEB-Pr phenotypes in several members of three apparently unrelated Danish families. A haplotype segregation study suggested a common ancestor in these kindred. Functional splicing...... shoulders. DEB-Pr is caused by either dominant (DDEB-Pr) or recessive mutations in the COL7A1 gene encoding type VII collagen (COLVII). The full spectrum of COL7A1 mutations in DEB-Pr remains elusive and the genotype-phenotype correlation is largely incomplete. Here, we report and functionally characterize...... analysis of the mutant exon by a COL7A1 minigene construct and computational prediction for splicing regulatory cis-sequences prove that the mutation alters the activity of an exonic splicing enhancer (ESE) critical for exon inclusion. These findings substantiate for the first time the involvement...

  20. Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

    Indian Academy of Sciences (India)

    MARYAM HAGHSHENAS; MOHAMMAD TAGHI AKBARI; SHOHREH ZARE KARIZI; FARAVAREH KHORDADPOOR DEILAMANI; SHAHRIAR NAFISSI; ZIVAR SALEHI

    2016-06-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progres-sive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletionsor duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to eval-uate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show anylarge deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependentprobe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 wassequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed fournonsense, one frameshift and two splice site mutations as well as two missense variants

  1. Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

    Science.gov (United States)

    Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

    2016-06-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

  2. Rodent-specific alternative exons are more frequent in rapidly evolving genes and in paralogs

    Directory of Open Access Journals (Sweden)

    Mironov Andrey A

    2009-06-01

    Full Text Available Abstract Background Alternative splicing is an important mechanism for generating functional and evolutionary diversity of proteins in eukaryotes. Here, we studied the frequency and functionality of recently gained, rodent-specific alternative exons. Results We projected the data about alternative splicing of mouse genes to the rat, human, and dog genomes, and identified exons conserved in the rat genome, but missing in more distant genomes. We estimated the frequency of rodent-specific exons while controlling for possible residual conservation of spurious exons. The frequency of rodent-specific exons is higher among predominantly skipped exons and exons disrupting the reading frame. Separation of all genes by the rate of sequence evolution and by gene families has demonstrated that rodent-specific cassette exons are more frequent in rapidly evolving genes and in rodent-specific paralogs. Conclusion Thus we demonstrated that recently gained exons tend to occur in fast-evolving genes, and their inclusion rate tends to be lower than that of older exons. This agrees with the theory that gain of alternative exons is one of the major mechanisms of gene evolution.

  3. Comparison of multiple vertebrate genomes reveals the birth and evolution of human exons.

    Science.gov (United States)

    Zhang, Xiang H-F; Chasin, Lawrence A

    2006-09-05

    Orthologous gene structures in eight vertebrate species were compared on a genomic scale to detect the birth and maturation of new internal exons during the course of evolution. We found that 40% of new human exons are alternatively spliced, and most of these are cassette exons (exons that are either included or skipped in their entirety) with low inclusion rates. This proportion decreases steadily as older and older exons are examined, even as splicing efficiency increases. Remarkably, the great majority of new cassette exons are composed of highly repeated sequences, especially Alu. Many new cassette exons are 5' untranslated exons; the proportion that code for protein increases steadily with age. New protein-coding exons evolve at a high rate, as evidenced by the initially high substitution rates (K(s) and K(a)), as well as the SNP density compared with older exons. This dynamic picture suggests that de novo recruitment rather than shuffling is the major route by which exons are added to genes, and that species-specific repeats could play a significant role in recent evolution.

  4. Stemcell Information: SKIP000182 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000182 ... Diseased GM23226 GM23226 ... 1型糖尿病 E10 diabetes (mellitus), juvenil...roblast. Multifactorial Defect 遺伝性若年発症I型糖尿病患者線維芽細胞(GM02416)由来iPS細胞 多因子の異常 human ES-like -- Retrovirus Oct4,

  5. Stemcell Information: SKIP000861 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000861 ... αβT αβT Normal 648B1 648B1 ... 30 30-39 Male Japanese Japanese -- No hiPSC Generat...ion from Peripheral Blood by using non-T medium. 末梢血由来iPS細胞。|T細胞の増殖には関与しない培地で作製。 hu

  6. Stemcell Information: SKIP000283 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000283 ... Diseased HPS0082 HPS0082 iPWS5 iPWS5 プラダー・ウィリー症候群 Q871 Prader Wil...patient of Prader Willi syndrome(RCB1560 PWS-Yamaguchi). del(15) was found in lymphocytes of the patient. 疾患特異的iPS細胞株。プラダー

  7. Stemcell Information: SKIP000262 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000262 ... Diseased GM23913 GM23913 ... フリードライヒ運動失調症 G111 Friedreich ataxia 22...9300 ... 30 30-39 Male ... Yes No iPS cell line derived from GM04078 fibroblast. Clinically affected with Friedrei...3 10.1016/j.stem.2010.09.014 Friedreich's ataxia induced pluripotent stem cells model intergenerational GAA-

  8. Stemcell Information: SKIP000625 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000625 ... Diseased HPS0171 HPS0171 ... デュシェンヌ型筋ジストロフィー G710 Duchenne Muscular...ent : Muscular dystrophy, Duchenne type. HPS0169 is derived from the same patient. ... デュシェンヌ型筋ジストロフィー患者由来iPS細胞

  9. Stemcell Information: SKIP000827 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000827 ... Diseased HPS0312 HPS0312 ... デュシェンヌ型筋ジストロフィー G710 ... Muscular dystrop...d from a muscular dystrophy, Duchenne type patient デュシェンヌ型筋ジストロフィー症患者由来iPS細胞株 human ES-like Research Grade R

  10. Stemcell Information: SKIP000618 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available o Disease specific iPS cell line derived from a muscular dystrophy, Duchenne type (manifestig carrier) patient デュシェンヌ... SKIP000618 ... Fibroblast derived from the skeletal muscle 骨格筋組織中の線維芽細胞 Gene carrier HPS0317 HPS0317 ... デュシェ...ンヌ型筋ジストロフィー G710 MUSCULAR DYSTROPHY, DUCHENNE TYPE 310200 ... -- Female ... No N

  11. Stemcell Information: SKIP000619 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available o Disease specific iPS cell line derived from a muscular dystrophy, Duchenne type (manifestig carrier) patient デュシェンヌ... SKIP000619 ... Fibroblast derived from the skeletal muscle 骨格筋組織中の線維芽細胞 Gene carrier HPS0322 HPS0322 ... デュシェ...ンヌ型筋ジストロフィー G710 Muscular dystrophy, Duchenne type 310200 ... -- Female ... No N

  12. Stemcell Information: SKIP000244 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000244 ... Diseased HPS0169 HPS0169 ... デュシェンヌ型筋ジストロフィー G710 Duchenne Muscular...ent : Muscular dystrophy, Duchenne type. HPS0171 is derived from the same patient. ... デュシェンヌ型筋ジストロフィー患者由来iPS細胞

  13. Stemcell Information: SKIP000330 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000330 ... mesenchymal stem cell 間葉系幹細胞 polydactylous human fingers 指 Normal Yub...ovation. 独立行政法人医薬基盤研究所JCRB細胞バンク http://cellbank.nibio.go.jp/~cellbank/cgi-bin/search_res_det.cgi?DB_NUM=1&ID=3377 ... ...621BMC Yub621BMC JCRB1113 JCRB1113 ... -- -- ... -- No JCRB1113 ... bone marrow-derived mesenchymal stem cell

  14. Stemcell Information: SKIP000792 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available tient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome QT 延長症候群 1 型患者由来iPS細胞 human ES-like... SKIP000792 ... Diseased LQT1 PII-2a LQT1 PⅡ-2a ... 遺伝性QT延長症候群1型 I45.81 long-QT syn

  15. Stemcell Information: SKIP000614 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000614 ... Diseased HPS0142 HPS0142 ... 家族性筋萎縮性側索硬化症 G122 Amyotrophic lateral sclerosis, Familia... a patient.Amyotrophic lateral sclerosis, Familial type. ... 疾患特異的iPS細胞株。家族性筋萎縮性側索硬化症患者由来。 human ES-like Resear

  16. Stemcell Information: SKIP000613 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000613 ... Diseased HPS0140 HPS0140 ... 家族性筋萎縮性側索硬化症 G122 Amyotrophic lateral sclerosis, Familia... a patient.Amyotrophic lateral sclerosis, Familial type. ... 疾患特異的iPS細胞株。家族性筋萎縮性側索硬化症患者由来。 human ES-like Resear

  17. Stemcell Information: SKIP001105 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001105 ... Diseased APP1E111 APP1E111 ... 家族性アルツハイマー病 G309 Alzheimer's disease... F693 ... -- -- ... Yes No Disease specific iPS cells from a patient of Alzheimer's disease. 疾患特異的iPS細胞株。APP-E693Δ変異家族性アルツハイマー

  18. Stemcell Information: SKIP000807 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000807 ... Diseased HPS0060 HPS0060 ... アルツハイマー型認知症 G30.9+ Dementia of Alzheim...tia of Alzheimers type. Order Form to RIKEN BRC (C-0042, C-0057, C-0007). 疾患特異的iPS細胞株。アルツハイマー型認知症患者由来。ガラス化法(

  19. Stemcell Information: SKIP000734 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000734 ... Diseased GM24675 GM24675 APPDp2 APPDp2 アルツハイマー病 G309 ALZHEIMER DI... hiPSC derived from fibroblast 家族性アルツハイマー病患者(APP遺伝子重複)維芽細胞由来iPS細胞 human ES-like Research Grade Retrovirus OC

  20. Stemcell Information: SKIP000791 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000791 ... Diseased 7643-32 7643-32 ... ティモシー症候群 I45.8 Timothy syndrome 601005... ... -- -- ... Yes No Human iPSCs.The Timothy syndrome iPSCs preserved the Timothy syndrome mutation. 疾患特異的iPS細胞。ティモシー症候群... investigate cardiac phenotypes in Timothy syndrome. Yazawa M, Hsueh B, Jia X, Pa

  1. A marginal level of dystrophin partially ameliorates hindlimb muscle passive mechanical properties in dystrophin-null mice.

    Science.gov (United States)

    Hakim, Chady H; Duan, Dongsheng

    2012-12-01

    The goal of this study was to determine whether a minimal level of dystrophin expression improves the passive mechanical properties of skeletal muscle in the murine Duchenne muscular dystrophy model. We compared the elastic and viscous properties of the extensor digitorum longus muscle (EDL) in mdx3cv and mdx4cv mice at 6, 14, and 20 months of age. Both strains are on the C57Bl/6 background, and both lose the full-length dystrophin protein. Interestingly, mdx3cv mice express a near full-length dystrophin at ≈ 5% of the normal level. We found that the stress-strain profile and the stress relaxation rate of the EDL in mdx3cv mice were partially preserved in all age groups compared with age-matched mdx4cv mice. Our results suggest that a low level of dystrophin expression may treat muscle stiffness in Duchenne muscular dystrophy. Copyright © 2012 Wiley Periodicals, Inc.

  2. Stemcell Information: SKIP000390 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000390 ... Diseased GM23764 GM23764 ... 統合失調症 F209 Schizophrenia 181500 ... ...より人工多能性幹細胞 (iPSC) を樹立。23回継代の凍結サンプル。同一人由来のリンパ球GM01490も参照。青年期から神経性無食欲症を発症。鬱<統合失調症。

  3. Stemcell Information: SKIP000272 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000272 ... Diseased HPS0129 HPS0129 ... 孤発性筋萎縮性側索硬化症 ... (classical, with UMN) G1...22 Amyotrophic lateral sclerosis, Sporadic type ... (classical, with UMN). ... -- -- Japanese Japanese N...o No Disease specific iPS cell line derived from a patient: Amyotrophic lateral sclerosis, Sporadic type (classical..., with UMN). ... 疾患特異的iPS細胞株。孤発性筋萎縮性側索硬化症 (classical, with UMN)患者由来。センダイウイルスベ

  4. Stemcell Information: SKIP000581 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000581 ... Diseased HCM 090909 HCM 090909 HCM 1 HCM 1 肥大型心筋症 I422 Hypertrophic cardiomyopathy...TPM1-Arg91Cys mutation that might be causal mutation of hypertrophic cardiomyopathy, by using four classic r... Endothelin-1 induces myofibrillar disarray and contractile vector variability in hypertrophic cardiomyopathy...ding (mouse) reprogramming factors constructed by the laboratory of Dr Yamanaka were obtained from Addgene. TPM1-Arg91Cys変異(肥大型心筋症

  5. Stemcell Information: SKIP000992 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000992 ... Diseased PARK8-LA11 PARK8-LA11 ... 遺伝性パーキンソン病:PARK8 G20 PARKINSON D...n's disease patient ... 遺伝性パーキンソン病患者由来iPS細胞 human ES-like Research Grade Lentivirus Klf4, Sox2, Oct4, c-Myc ... Ye...ISEASE 8, AUTOSOMAL DOMINANT; PARK8 607060 ... 66 60-69 Female ... Yes No iPS cells from familial Parkinso

  6. Stemcell Information: SKIP000993 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000993 ... Diseased PARK8-LB16 PARK8-LB16 ... 遺伝性パーキンソン病:PARK8 G20 PARKINSON D...n's disease patient ... 遺伝性パーキンソン病患者由来iPS細胞 human ES-like Research Grade Lentivirus Klf4, Sox2, Oct4, c-Myc ... Ye...ISEASE 8, AUTOSOMAL DOMINANT; PARK8 607060 ... 78 70-79 Female ... Yes No iPS cells from familial Parkinso

  7. Stemcell Information: SKIP000994 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000994 ... Diseased PARK8-LB21 PARK8-LB21 ... 遺伝性パーキンソン病:PARK8 G20 PARKINSON D...n's disease patient ... 遺伝性パーキンソン病患者由来iPS細胞 human ES-like Research Grade Lentivirus Klf4, Sox2, Oct4, c-Myc ... Ye...ISEASE 8, AUTOSOMAL DOMINANT; PARK8 607060 ... 78 70-79 Female ... Yes No iPS cells from familial Parkinso

  8. Stemcell Information: SKIP001095 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available line derived from a neonate.(1439 KURABO)| 新生児皮膚繊維芽細胞(1439 KURABO)由来iPS細胞 human ES-like Research ...on, Kyoto University 京都大学iPS細胞研究所 ... Information Only Center for iPS Cell Research and Application,Kyoto University 京都大学iPS細胞... SKIP001095 ... Normal WT-1-#1 WT-1-#1 ... 0-9 Male ... -- No Nomal human iPS cell...ling type II collagenopathy skeletal dysplasia by directed conversion and induced pluripotent stem cell

  9. Stemcell Information: SKIP000644 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000644 ... umbilical cord 臍帯(線維芽細胞) Normal HiPS-RIKEN-2D HiPS-RIKEN-2D ... ...bilical cord fibroblast, male) 臍帯由来線維芽細胞(RCB0197 HUC-Fm)iPS細胞. human ES-like Research Grade Retrovirus ... Oct3... ... Fetus Male Japanese Japanese -- No Human iPS cell line. Parent cell line of RCB0197 HUC-Fm(Normal um...010.00091.x Establishment of induced pluripotent stem cells from human neonatal tissues. Fujioka T, Shimizu

  10. Stemcell Information: SKIP001121 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available lines(C3) using transcription activator-like effector nuclease (TALEN; Fig. 3a ). TALENを用いてゲノム編集したiPS細胞... SKIP001121 ... Normal C-3-1-3M C-3-1-3M ... -- Female ... -- No Isogenic iPS cell...(C3) human ES-like Research Grade Plasmid ... -- ... Yes Differentiation of iPS cells into foreb...0.1038/nature13716 Synaptic dysregulation in a human iPS cell model of mental disorders. Wen Z, Nguyen HN, G

  11. Stemcell Information: SKIP000639 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000639 ... umbilical cord 臍帯(線維芽細胞) Normal HiPS-RIKEN-1D HiPS-RIKEN-1D ... ... umbilical cord fibroblast,Female) 臍帯由来線維芽細胞(RCB0436 HUC-F2)iPS細胞. human ES-like Research Grade Retrovirus O... ... Fetus Female Japanese Japanese -- No Human iPS cell line. Parent cell line of ... RCB0436 HUC-F2(Normal...774.2010.00091.x Establishment of induced pluripotent stem cells from human neonatal tissues. Fujioka T, Shi

  12. Stemcell Information: SKIP000949 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available inson disease 168006 ... 51 50-59 Female ... No No iPS cell line iPS細胞 human ES-like Research Grade Retrov... SKIP000949 ... dermal fibroblast 表皮繊維芽細胞 Diseased SP16.2 SP16.2 ... パーキンソン病 G20 Park...irus SOX2, KLF4 and OCT3/4 Yes Yes MEFs Lines of patient-specific iPS cells were maintained by mechanical di...ssociation of colonies and splitting 1:3 onto feeder cells in hESC medium or by l

  13. Stemcell Information: SKIP000953 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available inson disease 168006 ... 44 40-49 Male ... Yes No iPS cell line iPS細胞 human ES-like Research Grade Retrovi... SKIP000953 ... dermal fibroblast 表皮繊維芽細胞 Diseased SP06.1 SP06.1 ... パーキンソン病 G20 Park...rus SOX2, KLF4 and OCT3/4 Yes Yes MEFs Lines of patient-specific iPS cells were maintained by mechanical dis...sociation of colonies and splitting 1:3 onto feeder cells in hESC medium or by li

  14. Stemcell Information: SKIP000793 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ndrome type 1 192500 ... 42 40-49 Male ... Yes No Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome... SKIP000793 ... Diseased LQT1 PIII-2a LQT1 PⅢ-2a ... 遺伝性QT延長症候群1型 I45.81 long-QT sy... type1 QT 延長症候群 1 型患者由来iPS細胞 human ES-like Research Grade Retrovirus OCT3/4 SOX2 KLF4 c-MY

  15. Stemcell Information: SKIP000380 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ce-Moon(-Bardet)-Biedl Syndrome 209900 ... -- -- Japanese 日本人 No No Disease specific iPS cell line derived from Bardet-Biedl Syn...drome patient. HPS0156 is derived from the same patient. Bardet-Biedls症候群患者由来iPS細胞。疾患特異的iPS細胞株。バルデー·ビードル症候群... SKIP000380 ... Diseased HPS0157 HPS0157 ... ローレンス・ムーン症候群(バルデー・ビードル症候群) Q878 Lauren

  16. Stemcell Information: SKIP000794 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available drome type 2 613688 ... 28 20-29 Female ... Yes No Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome... SKIP000794 ... Diseased LQTS-hiPSCs LQTS-hiPSCs ... 遺伝性QT延長症候群2型 I45.8 long-QT syn... type2 QT 延長症候群 2 型患者由来iPS細胞 -- -- Retrovirus SOX-2, KLF-4, OCT-4 ... Yes MEF ES medium and

  17. Stemcell Information: SKIP000264 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ce-Moon(-Bardet)-Biedl Syndrome 209900 ... -- -- ... No No iPS cell line derived from Bardet-Biedl Syndrome... SKIP000264 ... Diseased HPS0156 HPS0156 ... ローレンス・ムーン症候群(バルデー・ビードル症候群) Q878 Lauren... patient. Bardet-Biedls症候群患者由来iPS細胞。| human ES-like -- Sendai virus SeV18+HS-OCT3/4/TSdeltaF, SeV18+HS-SOX

  18. Stemcell Information: SKIP000172 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000172 ... Diseased HPS0118 HPS0118 ... CINCA症候群 ... CINCA syndrome 607115 ... ... ... -- Male ... Yes No iPSC from CINCA syndrome patient with mutaion 1709A>G (Y570C) in NLRP3 as somatic mosaicism iPSC from CINCA syndr...12-03-417881 Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic m...ome(CINCA症候群) patient with mutaion 1709A>G (Y570C) in NLRP3 as somatic mosaicism hu

  19. Stemcell Information: SKIP000787 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000787 ... Diseased 7643-5 7643-5 ... ティモシー症候群 I45.8 Timothy syndrome 601005 ... ... ... -- -- ... Yes No Human iPSCs.The Timothy syndrome iPSCs preserved the Timothy syndrome mutation. 疾患特異的iPS細胞。ティモシー症候群...duced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome....--Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Yazawa M, Hs

  20. Stemcell Information: SKIP000105 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000105 ... Diseased A000180#2 A000180#2 ... エーラース・ダンロス症候群 Q796 Ehlers-Danlos syndrome... 130000 ... -- -- ... Yes No intractable disease-specific iPSC, Ehlers-Danlos syndrome, new varien...t 難病性疾患克服事業iPS細胞バンク 新型エーラース・ダンロス症候群 human ES-like -- Sendai virus Sendai Virus (DNAVEC: CytoTuneTM-iPS Cat.

  1. Stemcell Information: SKIP000790 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000790 ... Diseased 9862-61 9862-61 ... ティモシー症候群 I45.8 Timothy syndrome 601005... ... -- -- ... Yes No Human iPSCs.The Timothy syndrome iPSCs preserved the Timothy syndrome mutation. 疾患特異的iPS細胞。ティモシー症候群...induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome....--Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Yazawa M,

  2. Stemcell Information: SKIP000267 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000267 ... Diseased HPS0083 HPS0083 iCK2 iCK2 コケイン症候群 Q871 Cockayne's syndrome... 216400 ... -- Female japanese japanese No No Disease specific iPS cell line derived from a patient of Cockayne's syndrome.... コケイン症候群患者由来細胞(RCB0397 NCU-F10)に4因子(Oct3/4, Sox2, Klf4, c-Myc)を導入。|| human ES-like --

  3. Stemcell Information: SKIP000106 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000106 ... Diseased A000180#5 A000180#5 ... エーラース・ダンロス症候群 Q796 Ehlers-Danlos syndrome... 130000 ... -- -- ... Yes No intractable disease-specific iPSC, Ehlers-Danlos syndrome, new varien...t 難病性疾患克服事業iPS細胞バンク 新型エーラース・ダンロス症候群 human ES-like -- Sendai virus Sendai Virus (DNAVEC: CytoTuneTM-iPS Cat.

  4. Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry

    OpenAIRE

    Brown, Kristy J; Marathi, Ramya; Fiorillo, Alyson A; Ciccimaro, Eugene F.; Sharma, Seema; Rowlands, David S.; Rayavarapu, Sree; Nagaraju, Kanneboyina; Eric P. Hoffman; Hathout, Yetrib

    2012-01-01

    Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based ...

  5. Expression of dystrophin-glycoprotein complex at the skeletal muscle sarcolemma in Duchenne muscular dystrophy

    OpenAIRE

    Zhao, Lei; Chao-ping HU; Wang, Yi; Shui-zhen ZHOU; Shi, Yi-Yun; Xi-hua LI

    2015-01-01

    Background  Eccentric exercise or high tension exercise could cause damage to skeletal muscle structure, resulting in deficiency of dystrophin and secondary loss of dystrophin-glycoprotein complex (DGC) from the sarcolemma, which indicated that down-regulation of dystrophin was one of the key points of skeletal muscle injury from eccentric exercise. Duchenne muscular dystrophy (DMD) is caused by mutations of DMD gene, resulting in the absence of dystrophin, which means that skeletal muscles o...

  6. Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2.

    Science.gov (United States)

    Witting, N; Duno, M; Vissing, J

    2013-01-01

    Becker muscular dystrophy features progressive proximal weakness, wasting and often focal hypertrophy. We present a patient with pain and cramps from adolescence. Widespread muscle hypertrophy, preserved muscle strength and a 10-20-fold raised CPK were noted. Muscle biopsy was dystrophic, and Western blot showed a 95% reduction of dystrophin levels. Genetic analyses revealed a non-sense mutation in exon 2 of the dystrophin gene. This mutation is predicted to result in a Duchenne phenotype, but resulted in a mild Becker muscular dystrophy with widespread muscle hypertrophy. We suggest that this unusual phenotype is caused by translation re-initiation downstream from the mutation site. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Adolescent Breakfast Skipping: An Australian Study.

    Science.gov (United States)

    Shaw, Mary E.

    1998-01-01

    Reports on the findings of an Australian survey of adolescents concerning the extent of skipping breakfast. Finds that skippers are more likely to be dissatisfied with their body shape and to be on a diet to lose weight. Findings suggest that skipping breakfast is a matter of individual choice rather than a result of poverty. (Author/GCP)

  8. Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure.

    Science.gov (United States)

    Rafael, J A; Townsend, E R; Squire, S E; Potter, A C; Chamberlain, J S; Davies, K E

    2000-05-22

    The X-linked muscle wasting disease Duchenne muscular dystrophy is caused by the lack of dystrophin in muscle. Protein structure predictions, patient mutations, in vitro binding studies and transgenic and knockout mice suggest that dystrophin plays a mechanical role in skeletal muscle, linking the subsarcolemmal cytoskeleton with the extracellular matrix through its direct interaction with the dystrophin-associated protein complex (DAPC). Although a signaling role for dystrophin has been postulated, definitive data have been lacking. To identify potential non-mechanical roles of dystrophin, we tested the ability of various truncated dystrophin transgenes to prevent any of the skeletal muscle abnormalities associated with the double knockout mouse deficient for both dystrophin and the dystrophin-related protein utrophin. We show that restoration of the DAPC with Dp71 does not prevent the structural abnormalities of the post-synaptic membrane or the abnormal oxidative properties of utrophin/dystrophin-deficient muscle. In marked contrast, a dystrophin protein lacking the cysteine-rich domain, which is unable to prevent dystrophy in the mdx mouse, is able to ameliorate these abnormalities in utrophin/dystrophin-deficient mice. These experiments provide the first direct evidence that in addition to a mechanical role and relocalization of the DAPC, dystrophin and utrophin are able to alter both structural and biochemical properties of skeletal muscle. In addition, these mice provide unique insights into skeletal muscle fiber type composition.

  9. The roles of dystrophin and dystrobrevin : in synaptic signaling in drosophila

    NARCIS (Netherlands)

    Potikanond, Saranyapin

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a disease, characterized by progressive muscle wasting, caused by the lack of Dystrophin. A subset of DMD patients also have cognitive deficits likely due to the absence of Dystrophin from brain synapses where it is usually localized. Dystrophin and a number of o

  10. Stemcell Information: SKIP000186 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available s derived from GM03814(GMO3813'S mother) Fibroblast. SMN1 exons 7-8 deleted. Unaffected Coriell ID: GM23241 ... SMA1患者(GOM03813)母親線維芽細胞...038/nature07677 Stem Cell Model of Spinal Muscular Atrophy--Induced pluripotent stem cell

  11. Transcriptomic analysis of dystrophin RNAi knockdown reveals a central role for dystrophin in muscle differentiation and contractile apparatus organization

    Directory of Open Access Journals (Sweden)

    Graham Ian R

    2010-06-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystrophic cells and tissues suffer from non-specific changes and background noise due to heterogeneous comparisons and secondary pathologies. A study design in which a perfectly matched control cell population is used as reference for transcriptomic studies will give a much more specific insight into the effects of dystrophin deficiency and DMD pathophysiology. Results Using RNA interference (RNAi to knock down dystrophin in myotubes from C57BL10 mice, we created a homogenous model to study the transcriptome of dystrophin-deficient myotubes. We noted significant differences in the global gene expression pattern between these myotubes and their matched control cultures. In particular, categorical analyses of the dysregulated genes demonstrated significant enrichment of molecules associated with the components of muscle cell contractile unit, ion channels, metabolic pathways and kinases. Additionally, some of the dysregulated genes could potentially explain conditions and endophenotypes associated with dystrophin deficiency, such as dysregulation of calcium homeostasis (Pvalb and Casq1, or cardiomyopathy (Obscurin, Tcap. In addition to be validated by qPCR, our data gains another level of validity by affirmatively reproducing several independent studies conducted previously at genes and/or protein levels in vivo and in vitro. Conclusion Our results suggest that in striated muscles, dystrophin is involved in orchestrating proper development and organization of myofibers as contractile units, depicting a novel pathophysiology for DMD where the absence of dystrophin results in maldeveloped myofibers prone to physical stress and damage

  12. Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice.

    Directory of Open Access Journals (Sweden)

    Dejia Li

    Full Text Available Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv mice express a near-full length dystrophin protein at ∼5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.

  13. Disodium cromoglycate protects dystrophin-deficient muscle fibers from leakiness.

    Science.gov (United States)

    Marques, Maria Julia; Ventura Machado, Rafael; Minatel, Elaine; Santo Neto, Humberto

    2008-01-01

    In dystrophin-deficient fibers of mdx mice and in Duchenne dystrophy, the lack of dystrophin leads to sarcolemma breakdown and muscle degeneration. We verified that cromolyn, a mast-cell stabilizer agent, stabilized dystrophic muscle fibers using Evans blue dye as a marker of sarcolemma leakiness. Mdx mice (n=8; 14 days of age) received daily intraperitoneal injections of cromolyn (50 mg/kg body weight) for 15 days. Untreated mdx mice (n=8) were injected with saline. Cryostat cross-sections of the sternomastoid, tibialis anterior, and diaphragm muscles were stained with hematoxylin and eosin. Cromolyn dramatically reduced Evans blue dye-positive fibers in all muscles (P<0.05; Student's t-test) and led to a significant increase in the percentage of fibers with peripheral nuclei. This study supports the protective effects of cromolyn in dystrophic muscles and further indicates its action against muscle fiber leakiness in muscles that are differently affected by the lack of dystrophin.

  14. Deficiency of syntrophin, dystroglycan, and merosin in a female infant with a congenital muscular dystrophy phenotype lacking cysteine-rich and C-terminal domains of dystrophin.

    Science.gov (United States)

    Tachi, N; Ohya, K; Chiba, S; Matsuo, M; Patria, S Y; Matsumura, K

    1997-08-01

    Primary deficiency of merosin is the cause of the classic form of congenital muscular dystrophy (CMD) accompanied by brain white matter abnormalities. We report a female infant with dystrophinopathy who was deficient in merosin in skeletal muscle. The patient had a phenotype of typical CMD and white matter abnormalities on brain MRI. Merosin was greatly reduced in the biopsied skeletal muscle. However, the expression of dystroglycan and syntrophin was also greatly reduced, and the immunoreactivity for the antibodies against the cysteine-rich/C-terminal domains of dystrophin was absent in the sarcolemma. Reverse transcriptase polymerase chain reaction analysis of the dystrophin gene revealed a complete lack of exons 71 through 74. In skeletal muscle, only the mutant gene was expressed. These results suggest that the patient is a symptomatic Duchenne muscular dystrophy carrier with skewed X-inactivation. This patient illustrates for the first time that a dystrophin abnormality can cause a secondary deficiency of merosin in dystrophinopathy. The reduction of merosin may account for the clinical phenotype of CMD and correlate with the white matter abnormalities in our patient.

  15. Stemcell Information: SKIP001005 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001005 ... Normal ChiPSC18 ChiPSC18 Y00305 Y00305 ... 32 30-39 Male European.../North African European/North African -- No Cellartis human iPS cell line 18 (ChiPSC18)|Research Gra... ... Yes ... Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパ...AB Takara Bio Europe AB. タカラバイオヨーロッパAB Available Takara Bio Europe AB. タカラバイオヨーロッパAB http://www.clonte

  16. Stemcell Information: SKIP001001 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001001 ... Normal P11032 P11032 Y00225 Y00225 ... 38 30-39 Female European/North African Euro...F4,c-Myc ... -- ... Negative ... Yes ... Yes ... Yes G-banding Yes ... Yes ... Takara Bio Europe AB. タカラバイオヨーロッパ...AB Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパ...AB Not Available Takara Bio Europe AB. タカラバイオヨーロッパAB http://www.clontech.com/JP/Support/Contact_Technical_Support?sitex=10025:22372:US ... takarabio ...pean/North African -- No Cellartis human iPS cell line P11032|Research Grade(commer

  17. Stemcell Information: SKIP001006 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001006 ... Normal ChiPSC21 ChiPSC21 Y00315 Y00315 ... 26 20-29 Male European.../North African European/North African -- No Cellartis human iPS cell line 21 (ChiPSC21)|Research Gra... ... Yes ... Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパ...AB Takara Bio Europe AB. タカラバイオヨーロッパAB Available Takara Bio Europe AB. タカラバイオヨーロッパAB http://www.clonte

  18. Stemcell Information: SKIP001003 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001003 ... Normal ChiPSC7 ChiPSC7 Y00275 Y00275 ... 20 20-29 Female European.../North African European/North African -- No Cellartis human iPS cell line 7 (ChiPSC7)|Research Grade...es ... Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパ...AB Takara Bio Europe AB. タカラバイオヨーロッパAB Available Takara Bio Europe AB. タカラバイオヨーロッパAB http://www.clontech.c

  19. Stemcell Information: SKIP001004 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001004 ... Normal ChiPSC12 ChiPSC12 Y00285 Y00285 ... 24 20-29 Male European.../North African European/North African -- No Cellartis human iPS cell line 12 (ChiPSC12)|Research Gra... ... Yes ... Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパ...AB Takara Bio Europe AB. タカラバイオヨーロッパAB Available Takara Bio Europe AB. タカラバイオヨーロッパAB http://www.clonte

  20. Stemcell Information: SKIP001007 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001007 ... Normal ChiPSC22 ChiPSC22 Y00325 Y00325 ... 32 30-39 Male European.../North African European/North African -- No Cellartis human iPS cell line 22 (ChiPSC22)|Research Gra... ... Yes ... Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパAB Takara Bio Europe AB. タカラバイオヨーロッパ...AB Takara Bio Europe AB. タカラバイオヨーロッパAB Available Takara Bio Europe AB. タカラバイオヨーロッパAB http://www.clonte

  1. Stemcell Information: SKIP000583 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000583 ... Diseased HCM HJ HCM HJ HCM3 HCM3 肥大型心筋症 I422 Hypertrophic cardiomyopathy...ly999-Gln1004del mutation that is commonly causal mutation of hypertrophic cardiomyopathy, by using four sen...ndothelin-1 induces myofibrillar disarray and contractile vector variability in hypertrophic cardiomyopath...MYBPC3-Gly999-Gln1004del変異(肥大型心筋症の原因遺伝子の可能性をもつ)を持つ血液からiPS細胞を樹立した。4つのセンダイウイルスベクター(

  2. Stemcell Information: SKIP000543 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available l vectors pMXs-Oct4, pMXs-Sox2, pMXs-Klf4, and pMXs-c-Myc encoding (mouse) reprogramming factors. 健常人の皮膚細胞から...4つのレトロウイルスベクター(pMXs-Oct4, pMXs-Sox2, pMXs-Klf4, and pMXs-c-Mycを用いてiPS細胞を樹立した。 hum... SKIP000543 ... Normal iPS 090707 iPS 090707 ... -- -- ... -- No iPS cell...broblast (MEF) the standard hES cell medium (80% DMEM/F12, 20% KO Serum Replacement (Invitrogen) with 4 ng/m...y and contractile vector variability in hypertrophic cardiomyopathy-induced pluripotent stem cell-derived ca

  3. Stemcell Information: SKIP000504 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ate chip Chocolate chip ... Fetus Unknown ... -- No MRC-iPS-92|MRC5-derived iPS cells| MRC5由来iPS細胞| h... SKIP000504 ... fetal lung fibroblast 胎児肺線維芽細胞 Unknown MRC-iPS-92 MRC-iPS-92 Chocol...365-2443.2010.01459.x--10.1371/journal.pgen.1002085 Lectin microarray analysis of pluripotent and multipotent stem cell...s.--DNA methylation dynamics in human induced pluripotent stem cells over time. Toyoda M, Yamazak....nlm.nih.gov/pubmed/21155951--http://www.ncbi.nlm.nih.gov/pubmed/21637780 -- Some of clones of the same original cells have been used in the papers.

  4. Stemcell Information: SKIP000445 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available a Alameda ... Fetus Unknown ... -- No MRC-iPS-33|MRC5-derived iPS cells| MRC5由来iPS細胞| human ES-like R... SKIP000445 ... fetal lung fibroblast 胎児肺線維芽細胞 Unknown MRC-iPS-33 MRC-iPS-33 Alamed...01459.x--10.1371/journal.pgen.1002085 Lectin microarray analysis of pluripotent and multipotent stem cells.-...-DNA methylation dynamics in human induced pluripotent stem cells over time. Toyoda M, Yamazaki-Inoue M, Ita...ubmed/21155951--http://www.ncbi.nlm.nih.gov/pubmed/21637780 -- Some of clones of the same original cells have been used in the papers.

  5. Stemcell Information: SKIP001190 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001190 ... blood mononuclear cell 血中単核細胞 Diseased HPS0414 HPS0414 ... 原発性側索硬化症 G...m a patient :Primary lateral sclerosis (PLS). ... 原発性側索硬化症由来iPS細胞株。 human ES-like Research Grade Other Oct3/4, ...122 Primary Lateral Sclerosis ... -- -- Japanese 日本人 Yes No Disease specific iPS cell line derived fro...EN BioResource Center 理化学研究所 バイオリソースセンター ... Available RIKEN BioResource Center 理化学研究所 バイオリソースセンター ... http://www2.brc.riken.jp/lab/cell/detail.cgi?cell_no=HPS0414&type=1 ...

  6. Stemcell Information: SKIP001099 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available line derived from a neonate.(789013 KURABO)| 新生児皮膚繊維芽細胞(789013 KURABO)由来iPS細胞 human ES-like Res...ication, Kyoto University 京都大学iPS細胞研究所 ... Information Only Center for iPS Cell Research and Application,Kyoto University 京都大学iPS細胞... SKIP001099 ... Normal WT-2-#32 WT-2-#32 ... 0-9 Male ... -- No Nomal human iPS cell... Modeling type II collagenopathy skeletal dysplasia by directed conversion and in...duced pluripotent stem cells. Okada M, Ikegawa S, Morioka M, Yamashita A, Saito A, Sawai H, Murotsuki J, Oha

  7. Stemcell Information: SKIP000442 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available es Speckles ... Fetus Unknown ... -- No MRC-iPS-30|MRC5-derived iPS cells| MRC5由来iPS細胞| human ES-like... SKIP000442 ... fetal lung fibroblast 胎児肺線維芽細胞 Unknown MRC-iPS-30 MRC-iPS-30 Speckl...0.01459.x--10.1371/journal.pgen.1002085 Lectin microarray analysis of pluripotent and multipotent stem cells....--DNA methylation dynamics in human induced pluripotent stem cells over time. Toyoda M, Yamazaki-Inoue M, I.../pubmed/21155951--http://www.ncbi.nlm.nih.gov/pubmed/21637780 -- Some of clones of the same original cells have been used in the papers.

  8. Stemcell Information: SKIP000465 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available Smoky ... Fetus Unknown ... -- No MRC-iPS-53|MRC5-derived iPS cells| MRC5由来iPS細胞| human ES-like Resea... SKIP000465 ... fetal lung fibroblast 胎児肺線維芽細胞 Unknown MRC-iPS-53 MRC-iPS-53 Smoky ...9.x--10.1371/journal.pgen.1002085 Lectin microarray analysis of pluripotent and multipotent stem cells.--DNA... methylation dynamics in human induced pluripotent stem cells over time. Toyoda M, Yamazaki-Inoue M, Itakura...d/21155951--http://www.ncbi.nlm.nih.gov/pubmed/21637780 -- Some of clones of the same original cells have been used in the papers.

  9. Stemcell Information: SKIP000507 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ach Rorschach ... Fetus Unknown ... -- No MRC-iPS-96|MRC5-derived iPS cells| MRC5由来iPS細胞| human ES-li... SKIP000507 ... fetal lung fibroblast 胎児肺線維芽細胞 Unknown MRC-iPS-96 MRC-iPS-96 Rorsch...010.01459.x--10.1371/journal.pgen.1002085 Lectin microarray analysis of pluripotent and multipotent stem cell...s.--DNA methylation dynamics in human induced pluripotent stem cells over time. Toyoda M, Yamazaki-Inoue M,...ov/pubmed/21155951--http://www.ncbi.nlm.nih.gov/pubmed/21637780 -- Some of clones of the same original cells have been used in the papers.

  10. Stemcell Information: SKIP000469 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available Rufus ... Fetus Unknown ... -- No MRC-iPS-57|MRC5-derived iPS cells| MRC5由来iPS細胞| human ES-like Resea... SKIP000469 ... fetal lung fibroblast 胎児肺線維芽細胞 Unknown MRC-iPS-57 MRC-iPS-57 Rufus ...9.x--10.1371/journal.pgen.1002085 Lectin microarray analysis of pluripotent and multipotent stem cells.--DNA... methylation dynamics in human induced pluripotent stem cells over time. Toyoda M, Yamazaki-Inoue M, Itakura...d/21155951--http://www.ncbi.nlm.nih.gov/pubmed/21637780 -- Some of clones of the same original cells have been used in the papers.

  11. Stemcell Information: SKIP000287 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP000287 ... mesenchymal stem cell 間葉系幹細胞 Umbilical cord blood 臍帯血 Normal UCB408E7...ot Available National Institute of Biomedical Innovation. 独立行政法人医薬基盤研究所JCRB細胞バンク http://cellbank.nibio.go.jp/~cell...s with transgenic HPV E7. finite proliferation 臍帯血由来間葉系幹細胞 (有限増殖) fibroblast-like -- Retr...-32 UCB408E7-32 JCRB1109 JCRB1109 ... -- -- ... -- No JCRB1109 ... Human umbilical cord blood-derived mesenchymal stem cell...bank/cgi-bin/search_res_det.cgi?DB_NUM=1&ID=3373 ... 15647378 ... Immortalization of human fetal cell

  12. Stemcell Information: SKIP000382 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available alpha-Thalassemia, Mental Retardation Syndrome (ATR-X syndrome) 301040 ... -- -- ... No No Disease speci...fic iPS cell line derived from a patient : X-Linked alpha-Thalassemia, Mental Retardation Syndrome (ATR-X sy... SKIP000382 ... Diseased HPS0244 HPS0244 ... X連鎖αサラセミア·精神遅滞(ATR-X)症候群 D560 X-Linked...ndrome) 疾患特異的iPS細胞株。X連鎖alphaサラセミア·精神遅滞(ATR-X)症候群患者由来。 human ES-like Research Grad

  13. Stemcell Information: SKIP000388 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available l line derived from a patient :Alpha-Thalassemia/Mental Retardation Syndrome, X-Linked; ATRX. ... 疾患特異的iPS細胞株。X...alassemia/Mental Retardation Syndrome, X-Linked; ATRX 301040 ... -- -- ... No No Disease specific iPS cel... SKIP000388 ... Diseased HPS0207 HPS0207 ... X連鎖アルファ-サラセミア・精神遅滞(ATR-X)症候群 ... Alpha-Th...連鎖アルファ-サラセミア·精神遅滞(ATR-X)症候群患者由来。 human ES-like Research Grade Sendai virus SeV18+

  14. Stemcell Information: SKIP000876 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ient with F52L mutation in DCTN1. DCTN1にF52L遺伝子変異をもつPerry症候群患者1名から作製したiPS細胞 human E... SKIP000876 ... Diseased Perry syndrome patient iPSCs Perry syndrome patient iPSC...016.06.007 Cytoplasmic aggregates of dynactin in iPSC-derived tyrosine hydroxylase-positive neurons from a patient...s ... ペリー症候群 G20 Perry syndrome 168605 ... 61 60-69 Male ... Yes No ... Induced pluripotent stem cells (iPSCs) from a Perry syndrome pat

  15. The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

    Directory of Open Access Journals (Sweden)

    Ingrid E C Verhaart

    2014-01-01

    Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.

  16. Deletion analysis of the dystrophin gene in Duchenne and Becker muscular dystrophy patients: Use in carrier diagnosis

    Directory of Open Access Journals (Sweden)

    Kumari D

    2003-04-01

    Full Text Available The dystrophin gene was analyzed in 8 Duchenne muscular dystrophy (DMD and 10 Becker muscular dystrophy (BMD unrelated families (22 subjects: 18 index cases and 4 sibs for the presence of deletions by multiplex polymerase chain reaction (mPCR; 27 exons and Southern hybridization using 8 cDMD probes. Deletions were identified in 5 DMD and 7 BMD patients (6 index cases and 1 sib. The concordance between the clinical phenotype and 'reading frame hypothesis' was observed in 11/12 patients (92%. The female relatives of DMD/BMD patients with identifiable deletions were examined by quantitative mPCR. Carriers were identified in 7 families. We also describe a variation in the HindIII pattern with cDNA probe 8 and 11-14. Molecular characterization of the dystrophin gene in this study has been helpful in advising the patients concerning the inheritance of the condition, and carrier diagnosis of female relatives, and should also prove useful for prenatal diagnosis.

  17. Stemcell Information: SKIP000838 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ich deleated exon 6 and 7 in parkin gene.DNAVEC: SeV vectors, which were produced by DNAVEC Corp.(Cytotune). パーキンソン病患者T細胞より樹立したiPS細胞... ... 50-59 Male ... Yes No TPB27(DNAVEC) is iPSC line reprogrammed from a Parkinson disease patient's T-cells, wh

  18. Dystrophin, utrophin and {beta}-dystroglycan expression in skeletal muscle from patients with Becker muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Kawajiri, Masakazu; Mitsui, Takao; Kawai, Hisaomi [Univ. of Tokushima (Japan)] [and others

    1996-08-01

    The precise localization and semiquantitative correlation of dystrophin, utrophin and {beta}-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of {beta}-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of {beta}-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of {beta}-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to {beta}-dystroglycan. 35 refs., 3 figs., 1 tab.

  19. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

    Directory of Open Access Journals (Sweden)

    Charles W Sugnet

    2006-01-01

    Full Text Available Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.

  20. The dystrophin gene and cognitive function in the general population

    NARCIS (Netherlands)

    D. Vojinovic (Dina); H.H.H. Adams (Hieab); S. van der Lee (Sven); C.A. Ibrahim-Verbaas (Carla); R.W.W. Brouwer (Rutger); M.C.G.N. van den hout (Mirjam); E. Oole (Edwin); J. van Rooij (Jeroen); A.G. Uitterlinden (André); A. Hofman (Albert); W.F.J. van IJcken (Wilfred); A. Aartsma-Rus (Annemieke); G.-J.B. Van Ommen (Gert-Jan B.); M.A. Ikram (Arfan); C.M. van Duijn (Cornelia M.); N. Amin (Najaf)

    2015-01-01

    textabstractThe aim of our study is to investigate whether single-nucleotide dystrophin gene (DMD) variants associate with variability in cognitive functions in healthy populations. The study included 1240 participants from the Erasmus Rucphen family (ERF) study and 1464 individuals from the Rotterd

  1. Role of dystrophins and utrophins in platelet adhesion process.

    Science.gov (United States)

    Cerecedo, Doris; Mondragón, Ricardo; Cisneros, Bulmaro; Martínez-Pérez, Francisco; Martínez-Rojas, Dalila; Rendón, Alvaro

    2006-07-01

    Platelets are crucial at the site of vascular injury, adhering to the sub-endothelial matrix through receptors on their surface, leading to cell activation and aggregation to form a haemostatic plug. Platelets display focal adhesions as well as stress fibres to contract and facilitate expulsion of growth and pro-coagulant factors contained in the granules and to constrict the clot. The interaction of F-actin with different actin-binding proteins determines the properties and composition of the focal adhesions. Recently, we demonstrated the presence of dystrophin-associated protein complex corresponding to short dystrophin isoforms (Dp71d and Dp71) and the uthophin gene family (Up400 and Up71), which promote shape change, adhesion, aggregation, and granule centralisation. To elucidate participation of both complexes during the platelet adhesion process, their potential association with integrin beta-1 fraction and the focal adhesion system (alpha-actinin, vinculin and talin) was evaluated by immunofluorescence and immunoprecipitation assays. It was shown that the short dystrophin-associated protein complex participated in stress fibre assembly and in centralisation of cytoplasmic granules, while the utrophin-associated protein complex assembled and regulated focal adhesions. The simultaneous presence of dystrophin and utrophin complexes indicates complementary structural and signalling mechanisms to the actin network, improving the platelet haemostatic role.

  2. Ocular and neurodevelopmental features of Duchenne muscular dystrophy: a signature of dystrophin function in the central nervous system.

    Science.gov (United States)

    Ricotti, Valeria; Jägle, Herbert; Theodorou, Maria; Moore, Anthony T; Muntoni, Francesco; Thompson, Dorothy A

    2016-04-01

    Multiple isoforms of dystrophin (Dp427, Dp260, Dp140, Dp71) are expressed differentially in the central nervous system (CNS) including the retinal layers. Disruption of these protein products is responsible for cognitive dysfunction, electroretinogram (ERG) abnormalities and behavioural disorders in Duchenne muscular dystrophy (DMD). We studied the ocular characteristics and neuropsychiatric profile of 16 DMD boys. The ISCEV standard, full-field flash ERGs were assessed. Intellectual ability and behavioural disturbances were measured. All genotypes were associated with mildly abnormal photopic ERG a:b-wave amplitude ratios. In addition, we identified the following genotype/phenotype correlations: boys with mutations upstream of exon 30 (ie, isolated Dp427 altered expression) showed normal scotopic a:b ratios, abnormal photopic oscillatory potential OP2 and normal scotopic OP2. Conversely, all boys with DMD mutations downstream of exon 30 showed profoundly 'negative' scotopic ERGs (a:b ratios >1). In these patients, the involvement of Dp260 isoform resulted in the absence of slow rod pathway signalling in15 Hz scotopic flicker ERGs. These boys had abnormal scotopic OP2 and normal photopic OP2. Finally, children with mutations also affecting Dp71 were associated with more pronounced electronegative ERGs. When correlating ERGs to neurodevelopmental outcome, we found a positive correlation between negative scotopic ERGs and neurodevelopmental disturbances, and the most severe findings were in boys with Dp71 disruption. These findings suggest a strong association between DMD mutations affecting different DMD isoforms with characteristically abnormal scotopic ERGs and severe neurodevelopmental problems. The role of the ERG as a potential biomarker for dystrophin function in the CNS and response to novel genetic therapies warrants further exploration.

  3. Antisense Oligonucleotide-mediated Exon Skipping as a Systemic Therapeutic Approach for Recessive Dystrophic Epidermolysis Bullosa : Exon Skipping as Systemic Therapy for RDEB

    NARCIS (Netherlands)

    Bremer, Jeroen; Bornert, Olivier; Nyström, Alexander; Gostynski, Antoni; Jonkman, Marcel F; Aartsma-Rus, Annemieke; van den Akker, Peter C; Pasmooij, Anna MG

    2016-01-01

    The "generalized severe" form of recessive dystrophic epidermolysis bullosa (RDEB-gen sev) is caused by bi-allelic null mutations in COL7A1, encoding type VII collagen. The absence of type VII collagen leads to blistering of the skin and mucous membranes upon the slightest trauma. Because most patie

  4. Stemcell Information: SKIP000680 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ions in the ratio 3:1:1:1:1. 胎児肺線維芽細胞由来iPS細胞。|低酸素下でKlf4, c-Myc, Oct4, Sox2とLIN28を...3:1:1:1:1の割合でコードした合成RNAを、線維芽細胞株に20日間毎日添加し、遺伝子を細胞へ導入した。 human ES-like Research Grade Other OCT4 , SOX2 , KLF4... SKIP000680 ... Lung Lung Normal MRC5-RiPS-1.11 MRC5-RiPS-1.11 ... Fetus Male ... -- No hiPS-cell...s derived from human fetal lung fibroblast cells (MRC-5 Line).They were derived using...on ... 20888316 10.1016/j.stem.2010.08.012 Highly efficient reprogramming to pluripotency and directed differentiation of human cell

  5. Stemcell Information: SKIP001102 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available SKIP001102 ... induced chondrogenic (iChon) cells ダイレクトリプログラミングで作製した軟骨細胞 ... Normal WT...n Dermal Fibroblasts(789013 KURABO). 新生児皮膚繊維芽細胞(789013 KURABO)をダイレクトリプログラミングにより軟骨細胞へと誘導(iChon細胞) Other Resea...n, Kyoto University 京都大学iPS細胞研究所 ... Information Only Center for iPS Cell Researc...h and Application,Kyoto University 京都大学iPS細胞研究所 CiRA https://www.cira.kyoto-u.ac.jp/e/index.html ... 2518757...-2-iChon WT-2-iChon ... 0-9 Male ... -- No Direct iInduction of Chondrogenic(iChon) cells from Huma

  6. Stemcell Information: SKIP000221 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available N-2A HPS0009 HPS0009 ... -- Male ... -- No Human iPS cell line. ヒトiPS細胞。臍帯由来線維芽細胞(RCB0197 HUC-Fm)由来。 ... SKIP000221 ... Umbilical cord(Fibroblast) 臍帯(線維芽細胞) Normal HiPS-RIKEN-2A HiPS-RIKE...human ES-like -- Retrovirus Retroviral vector pMXs, Oct3/4, Sox2, Klf4, c-Myc ... Yes MEF (X-rays:5000R or MMC) 1-1.5x10^(6) cell.../www2.brc.riken.jp/lab/cell/detail.cgi?cell_no=HPS0009&type=1 ... ...学研究所バイオリソースセンター RIKEN BRC 理化学研究所バイオリソースセンター Yukio Nakamura 中村幸夫 Available RIKEN BRC 理化学研究所バイオリソースセンター http:/

  7. Stemcell Information: SKIP000677 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ns in the ratio 3:1:1:1:1. 胎児肺線維芽細胞由来iPS細胞。|低酸素下でKlf4, c-Myc, Oct4, Sox2とLIN28を3:...1:1:1:1の割合でコードした合成RNAを、線維芽細胞株に20日間毎日添加し、遺伝子を細胞へ導入した。 human ES-like Research Grade Other OCT4 , SOX2 , KLF4 ,... SKIP000677 ... Lung Lung Normal MRC5-RiPS-1.2 MRC5-RiPS-1.2 ... Fetus Male ... -- No hiPS-cell...s derived from human fetal lung fibroblast cells (MRC-5 Line).They were derived using m...8.012 Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthe

  8. Stemcell Information: SKIP000679 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ns in the ratio 3:1:1:1:1. 胎児肺線維芽細胞由来iPS細胞。|低酸素下でKlf4, c-Myc, Oct4, Sox2とLIN28を3:...1:1:1:1の割合でコードした合成RNAを、線維芽細胞株に20日間毎日添加し、遺伝子を細胞へ導入した。 human ES-like Research Grade Other OCT4 , SOX2 , KLF4 ,... SKIP000679 ... Lung Lung Normal MRC5-RiPS-1.9 MRC5-RiPS-1.9 ... Fetus Male ... -- No hiPS-cell...s derived from human fetal lung fibroblast cells (MRC-5 Line).They were derived using m....1016/j.stem.2010.08.012 Highly efficient reprogramming to pluripotency and directed differentiation of human cell

  9. Reading Skill and Word Skipping: Implications for Visual and Linguistic Accounts of Word Skipping

    Science.gov (United States)

    Eskenazi, Michael A.; Folk, Jocelyn R.

    2015-01-01

    We investigated whether high-skill readers skip more words than low-skill readers as a result of parafoveal processing differences based on reading skill. We manipulated foveal load and word length, two variables that strongly influence word skipping, and measured reading skill using the Nelson-Denny Reading Test. We found that reading skill did…

  10. Reading Skill and Word Skipping: Implications for Visual and Linguistic Accounts of Word Skipping

    Science.gov (United States)

    Eskenazi, Michael A.; Folk, Jocelyn R.

    2015-01-01

    We investigated whether high-skill readers skip more words than low-skill readers as a result of parafoveal processing differences based on reading skill. We manipulated foveal load and word length, two variables that strongly influence word skipping, and measured reading skill using the Nelson-Denny Reading Test. We found that reading skill did…

  11. Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies

    Directory of Open Access Journals (Sweden)

    Caroline Perronnet

    2010-01-01

    Full Text Available Two decades of molecular, cellular, and functional studies considerably increased our understanding of dystrophins function and unveiled the complex etiology of the cognitive deficits in Duchenne muscular dystrophy (DMD, which involves altered expression of several dystrophin-gene products in brain. Dystrophins are normally part of critical cytoskeleton-associated membrane-bound molecular scaffolds involved in the clustering of receptors, ion channels, and signaling proteins that contribute to synapse physiology and blood-brain barrier function. The utrophin gene also drives brain expression of several paralogs proteins, which cellular expression and biological roles remain to be elucidated. Here we review the structural and functional properties of dystrophins and utrophins in brain, the consequences of dystrophins loss-of-function as revealed by numerous studies in mouse models of DMD, and we discuss future challenges and putative therapeutic strategies that may compensate for the cognitive impairment in DMD based on experimental manipulation of dystrophins and/or utrophins brain expression.

  12. Dystrophin Distribution and Expression in Human and Experimental Temporal Lobe Epilepsy

    Science.gov (United States)

    Hendriksen, Ruben G. F.; Schipper, Sandra; Hoogland, Govert; Schijns, Olaf E. M. G.; Dings, Jim T. A.; Aalbers, Marlien W.; Vles, Johan S. H.

    2016-01-01

    Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE). Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats. Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls. Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated

  13. Skip segment Hirschsprung's disease: a systematic review.

    LENUS (Irish Health Repository)

    O'Donnell, Anne-Marie

    2012-02-01

    PURPOSE: Hirschsprung\\'s disease is characterised by the congenital absence of ganglion cells beginning in the distal rectum and extending proximally for varying distances. \\'Zonal aganglionosis\\' is a phenomenon involving a zone of aganglionosis occurring within normally innervated intestine. \\'Skip segment\\' Hirschsprung\\'s disease (SSHD) involves a \\'skip area\\' of normally ganglionated intestine, surrounded proximally and distally by aganglionosis. While Hirschsprung\\'s disease is believed to be the result of incomplete craniocaudal migration of neural crest-derived cells, the occurrence of SSHD has no clear embryological explanation. The aim of this study was to perform a systematic review of SSHD, reported in the literature between 1954 and 2009, in order to determine the clinical characteristics of this rare entity and its significance. METHODS: The first reported case of SSHD was published in 1954. A systematic review of SSHD cases in the literature, from 1954 to 2009, was carried out using the electronic database \\'Pubmed\\'. Detailed information was recorded regarding the age, gender, presenting symptoms and location of the skip segment in each patient. RESULTS: 24 cases of SSHD have been reported in the literature to date. 18\\/24 (75%) of these cases were males and 6\\/24 (25%) were females. Of these, 22\\/24 (92%) were cases of total colonic aganglionosis (TCA), and 2\\/24 (8%) were rectosigmoid Hirschsprung\\'s disease. Of the 22 TCA cases, 9 (41%) had a skip segment in the transverse colon, 6 (27%) in the ascending colon, 2 (9%) in the caecum and 5 (23%) had multiple skip segments. In both rectosigmoid Hirschsprung\\'s disease cases, the skip segment was in the sigmoid colon. Overall, the length of the skip segment was variable, with the entire transverse colon ganglionated in some cases. CONCLUSION: SSHD occurs predominantly in patients with TCA. The existence of a skip area of normally innervated colon in TCA may influence surgical

  14. Evolutionary study of vertebrate and invertebrate members of the dystrophin and utrophin gene family

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, R.G.; Nicholson, L.; Bobrow, M. [Paediatric Research Unit, London (United Kingdom)] [and others

    1994-09-01

    Vertebrates express two members of the dystrophin gene family. The prototype, dystrophin, is expressed in muscle and neural tissue, and is defective in the human disorders Duchenne and Becker muscular dystrophy (DMD, BMD). The dystrophin homologue utrophin is more generally expressed but has not yet been associated with a genetic disorder. The function of neither protein is clear. A comparison of human utrophin with the known dystrophins (human, mouse, chicken, Torpedo) suggests that dystrophin and utrophin diverged before the vertebrate radiation. We have used reverse-transcript PCR (RT-PCR) directed by degenerate primers to characterize dystrophin and utrophin transcripts from a range of vertebrate and invertebrate animals. Our results suggest that the duplication leading to distinct dystrophin and utrophin genes occurred close to the point of divergence of urochordates from the cephalochordate-vertebrate lineage. This divergence may have occurred to fulfill a novel role which arose at this point, or may reflect a need for separate regulation of the neuromuscular and other functions of the ancient dystrophin. Our data include sequences of the first non-human utrophins to be characterized, and show these to be substantially more divergent than their cognate dystrophins. In addition, our results provide a large body of information regarding the tolerance of amino acid positions in the cysteine-rich and C-terminal domains to substitution. This will aid the interpretations of DMD and BMD missense mutations in these regions.

  15. Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

    Directory of Open Access Journals (Sweden)

    Eric K Johnson

    Full Text Available Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

  16. Evolutionary history of exon shuffling.

    Science.gov (United States)

    França, Gustavo S; Cancherini, Douglas V; de Souza, Sandro J

    2012-06-01

    Exon shuffling has been characterized as one of the major evolutionary forces shaping both the genome and the proteome of eukaryotes. This mechanism was particularly important in the creation of multidomain proteins during animal evolution, bringing a number of functional genetic novelties. Here, genome information from a variety of eukaryotic species was used to address several issues related to the evolutionary history of exon shuffling. By comparing all protein sequences within each species, we were able to characterize exon shuffling signatures throughout metazoans. Intron phase (the position of the intron regarding the codon) and exon symmetry (the pattern of flanking introns for a given exon or block of adjacent exons) were features used to evaluate exon shuffling. We confirmed previous observations that exon shuffling mediated by phase 1 introns (1-1 exon shuffling) is the predominant kind in multicellular animals. Evidence is provided that such pattern was achieved since the early steps of animal evolution, supported by a detectable presence of 1-1 shuffling units in Trichoplax adhaerens and a considerable prevalence of them in Nematostella vectensis. In contrast, Monosiga brevicollis, one of the closest relatives of metazoans, and Arabidopsis thaliana, showed no evidence of 1-1 exon or domain shuffling above what it would be expected by chance. Instead, exon shuffling events are less abundant and predominantly mediated by phase 0 introns (0-0 exon shuffling) in those non-metazoan species. Moreover, an intermediate pattern of 1-1 and 0-0 exon shuffling was observed for the placozoan T. adhaerens, a primitive animal. Finally, characterization of flanking intron phases around domain borders allowed us to identify a common set of symmetric 1-1 domains that have been shuffled throughout the metazoan lineage.

  17. Autologous skeletal muscle derived cells expressing a novel functional dystrophin provide a potential therapy for Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Meng, Jinhong; Counsell, John R; Reza, Mojgan; Laval, Steven H; Danos, Olivier; Thrasher, Adrian; Lochmüller, Hanns; Muntoni, Francesco; Morgan, Jennifer E

    2016-01-27

    Autologous stem cells that have been genetically modified to express dystrophin are a possible means of treating Duchenne Muscular Dystrophy (DMD). To maximize the therapeutic effect, dystrophin construct needs to contain as many functional motifs as possible, within the packaging capacity of the viral vector. Existing dystrophin constructs used for transduction of muscle stem cells do not contain the nNOS binding site, an important functional motif within the dystrophin gene. In this proof-of-concept study, using stem cells derived from skeletal muscle of a DMD patient (mdcs) transplanted into an immunodeficient mouse model of DMD, we report that two novel dystrophin constructs, C1 (ΔR3-R13) and C2 (ΔH2-R23), can be lentivirally transduced into mdcs and produce dystrophin. These dystrophin proteins were functional in vivo, as members of the dystrophin glycoprotein complex were restored in muscle fibres containing donor-derived dystrophin. In muscle fibres derived from cells that had been transduced with construct C1, the largest dystrophin construct packaged into a lentiviral system, nNOS was restored. The combination of autologous stem cells and a lentivirus expressing a novel dystrophin construct which optimally restores proteins of the dystrophin glycoprotein complex may have therapeutic application for all DMD patients, regardless of their dystrophin mutation.

  18. Power inverter implementing phase skipping control

    Science.gov (United States)

    Somani, Utsav; Amirahmadi, Ahmadreza; Jourdan, Charles; Batarseh, Issa

    2016-10-18

    A power inverter includes a DC/AC inverter having first, second and third phase circuitry coupled to receive power from a power source. A controller is coupled to a driver for each of the first, second and third phase circuitry (control input drivers). The controller includes an associated memory storing a phase skipping control algorithm, wherein the controller is coupled to receive updating information including a power level generated by the power source. The drivers are coupled to control inputs of the first, second and third phase circuitry, where the drivers are configured for receiving phase skipping control signals from the controller and outputting mode selection signals configured to dynamically select an operating mode for the DC/AC inverter from a Normal Control operation and a Phase Skipping Control operation which have different power injection patterns through the first, second and third phase circuitry depending upon the power level.

  19. The Relation between Breakfast Skipping and School Performance in Adolescents

    Science.gov (United States)

    Boschloo, Annemarie; Ouwehand, Carolijn; Dekker, Sanne; Lee, Nikki; de Groot, Renate; Krabbendam, Lydia; Jolles, Jelle

    2012-01-01

    Breakfast skipping is common in adolescents, but research on the effects of breakfast skipping on school performance is scarce. This current cross-sectional survey study of 605 adolescents aged 11-18 years investigated whether adolescents who habitually skip breakfast have lower end-of-term grades than adolescents who eat breakfast daily.…

  20. Stemcell Information: SKIP000206 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available kin遺伝子のexon毎に設計したprimerを用いてgenomic PCR解析を行った。|      その結果、患者由来線維芽細胞およびPA1.9.22iPSにおいてExon2,3,4のhomozygous del... : ICC法| Tra-1-81 : ICC法||染色体解析 : 実施 CGH array解析済み。PARK2遺伝子の欠損以外、変異は見当たらなかった。|胚葉体...形成実験 : 実施 胚葉体形成確認|テラトーマ形成実験 : 実施 テラトーマ形成確認|in vitro分化能解析 : 実施 神経への分化確認|マイコプラズマ汚染検査 : 実施 陰性|細胞同定検査(STR多型解析) : 未実施|クローニング : ||その他の特性情報 : 樹立に用いた外来遺伝子の発現抑制を確認した。

  1. Stemcell Information: SKIP000207 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available arkin遺伝子のexon毎に設計したprimerを用いてgenomic PCR解析を行った。|      その結果、患者由来線維芽細胞およびPA1.9.22iPSにおいてExon2,3,4のhomozygous d...60 : ICC法| Tra-1-81 : ICC法||染色体解析 : 実施 CGH array解析済み。PARK2遺伝子の欠損以外、変異は見当たらなかった。|胚...葉体形成実験 : 実施 胚葉体形成確認|テラトーマ形成実験 : 実施 テラトーマ形成確認|in vitro分化能解析 : 実施 神経への分化確認|マイコプラズマ汚染検査 : 実施 陰性|細胞同定検査(STR多型解析) : 未実施|クローニング : ||その他の特性情報 : 樹立に用いた外来遺伝子の発現抑制を確認した。

  2. Stemcell Information: SKIP000521 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available kin遺伝子のexon毎に設計したprimerを用いてgenomic PCR解析を行った。|      その結果、患者由来線維芽細胞およびPA1.9.22iPSにおいてExon2,3,4のhomozygous del... : ICC法| Tra-1-81 : ICC法||染色体解析 : 実施 CGH array解析済み。PARK2遺伝子の欠損以外、変異は見当たらなかった。|胚葉体...形成実験 : 実施 胚葉体形成確認|テラトーマ形成実験 : 実施 テラトーマ形成確認|in vitro分化能解析 : 実施 神経への分化確認|マイコプラズマ汚染検査 : 実施 陰性|細胞同定検査(STR多型解析) : 未実施|クローニング : ||その他の特性情報 : 樹立に用いた外来遺伝子の発現抑制を確認した。

  3. EasyExonPrimer: automated primer design for exon sequences.

    Science.gov (United States)

    Wu, Xiaolin; Munroe, David J

    2006-01-01

    EasyExonPrimer is a web-based software that automates the design of PCR primers to amplify exon sequences from genomic DNA. EasyExonPrimer is written in Perl and uses Primer3 to design PCR primers based on the genome builds and annotation databases available at the University of California, Santa Cruz (UCSC) Genome Browser database (http://genome.ucsc.edu/). It masks repeats and known single nucleotide polymorphism (SNP) sites in the genome and designs standardised primers using optimised conditions. Users can input genes by RefSeq mRNA ID, gene name or keyword. The primer design is optimised for large-scale resequencing of exons. For exons larger than 1 kb, the user has the option of breaking the exon sequence down into overlapping smaller fragments. All primer pairs are then verified using the In-Silico PCR software to test for uniqueness in the genome. We have designed >1000 pairs of primers for 90 genes; 95% of the primer pairs successfully amplified exon sequences under standard PCR conditions without requiring further optimisation. EasyExonPrimer is available from http://129.43.22.27/~primer/. The source code is also available upon request. Xiaolin Wu (forestwu@mail.nih.gov).

  4. DESIGN OF OPTIMAL CARRY SKIP ADDER AND CARRY SKIP BCD ADDER USING REVERSIBLE LOGIC GATES

    OpenAIRE

    Praveena Murugesan; Thanushkodi Keppanagounder

    2014-01-01

    Reversible logic circuits have the ability to produce zero power dissipation which has found its importance in quantum computing, optical computing and low power digital circuits. The study presents improved and efficient reversible logic circuits for carry skip adder and carry skip BCD adder. The performance of the proposed architecture is better than the existing works in terms of gate count, garbage outputs and constant inputs. This design forms the basis for different quantum ALU and embe...

  5. The influence of low dystrophin levels on disease pathology in mouse models for Duchenne Muscular Dystrophy

    NARCIS (Netherlands)

    Putten, Maaike van

    2013-01-01

    Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive to exercise-induced damage, resulting in progressive muscle wasting, loss of ambulation and premature de

  6. Carrier detection of duchenne and becker muscular dystrophy using muscle dystrophin immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Acary S. Bulle Oliveira

    1992-12-01

    Full Text Available To ascertain whether dystrophin immunohistochemistry could improve DMD/ BMD carrier detection, we analyzed 14 muscle biopsies from 13 DMD and one BMD probable and possible carriers. All women were also evaluated using conventional methods, including genetic analysis, clinical and neurological evaluation, serum CK levels, KMG, and muscle biopsy. In 6 cases, there was a mosaic of dystrophin-positive and dystrophin-deficient fibers that allowed to make the diagnosis of a carrier state. Comparing dystrophin immunohistochemistry to the traditional methods, it was noted that this method is less sensitive than serum CK measuremens, but is more sensitive than EMG and muscle biopsy. The use of dystrophin immunohistochemistry in addition to CK, EMG and muscle biopsy improved the accuracy of carrier detection. This method is also helpful to distinguish manifesting DMD carriers from patients with other neuromuscular diseases like limb-girdle muscular dystrophy and spinal muscular atrophy.

  7. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity.

    Science.gov (United States)

    Cox, G A; Cole, N M; Matsumura, K; Phelps, S F; Hauschka, S D; Campbell, K P; Faulkner, J A; Chamberlain, J S

    1993-08-19

    Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked recessive diseases caused by defective expression of dystrophin. The mdx mouse, an animal model for DMD, has a mutation that eliminates expression of the 427K muscle and brain isoforms of dystrophin. Although these animals do not display overt muscle weakness or impaired movement, the diaphragm muscle of the mdx mouse is severely affected and shows progressive myofibre degeneration and fibrosis which closely resembles the human disease. Here we explore the feasibility of gene therapy for DMD by examining the potential of a full-length dystrophin transgene to correct dystrophic symptoms in mdx mice. We find that expression of dystrophin in muscles of transgenic mdx mice eliminates the morphological and immunohistological symptoms of muscular dystrophy. In addition, overexpression of dystrophin prevents the development of the abnormal mechanical properties associated with dystrophic muscle without causing deleterious side effects. Our results provide functional evidence for the feasibility of gene therapy for DMD.

  8. Correlates of meal skipping in young adults: a systematic review.

    Science.gov (United States)

    Pendergast, Felicity J; Livingstone, Katherine M; Worsley, Anthony; McNaughton, Sarah A

    2016-12-01

    Meal skipping rates may be highest during young adulthood, a period of transition and development. Although these dietary behaviours may increase future risk of chronic disease, limited research has investigated correlates of meal skipping in young adults. A systematic literature search was conducted to identify studies that investigated correlates of meal skipping behaviours in young adults (aged 18-30 years). EBSCO host, MEDLINE Complete, Global Health, Scopus, EMBASE, Web of Science and Informit platforms were searched for eligible articles. Correlates were defined as any factor that was either associated with meal skipping or was self-reported by the participant to have an influence on meal skipping. Randomised controlled trials, prospective cohort studies, case-control studies, nested case-control studies, cross-sectional studies, and longitudinal studies were eligible for inclusion. Three-hundred and thirty-one articles were identified, 141 full-text articles assessed for eligibility, resulting in 35 included studies. Multiple methodological and reporting weaknesses were apparent in the reviewed studies with 28 of the 35 studies scoring a negative rating in the risk of bias assessment. Meal skipping (any meal), defined as the skipping of any meal throughout the day, was reported in 12 studies with prevalence ranging between 5 and 83%. The remaining 25 studies identified specific meals and their skipping rates, with breakfast the most frequently skipped meal 14-88% compared to lunch 8-57% and dinner 4-57%. Lack of time was consistently reported as an important correlate of meal skipping, compared with correlates such as cost and weight control, while sex was the most commonly reported associated correlate. Breakfast skipping was more common among men while lunch or dinner skipping being more common among women. This review is the first to examine potential correlates of meal skipping in young adults. Future research would benefit from stronger design and

  9. Role of dystrophin in airway smooth muscle phenotype, contraction and lung function.

    Directory of Open Access Journals (Sweden)

    Pawan Sharma

    Full Text Available Dystrophin links the transmembrane dystrophin-glycoprotein complex to the actin cytoskeleton. We have shown that dystrophin-glycoprotein complex subunits are markers for airway smooth muscle phenotype maturation and together with caveolin-1, play an important role in calcium homeostasis. We tested if dystrophin affects phenotype maturation, tracheal contraction and lung physiology. We used dystrophin deficient Golden Retriever dogs (GRMD and mdx mice vs healthy control animals in our approach. We found significant reduction of contractile protein markers: smooth muscle myosin heavy chain (smMHC and calponin and reduced Ca2+ response to contractile agonist in dystrophin deficient cells. Immunocytochemistry revealed reduced stress fibers and number of smMHC positive cells in dystrophin-deficient cells, when compared to control. Immunoblot analysis of Akt1, GSK3β and mTOR phosphorylation further revealed that downstream PI3K signaling, which is essential for phenotype maturation, was suppressed in dystrophin deficient cell cultures. Tracheal rings from mdx mice showed significant reduction in the isometric contraction to methacholine (MCh when compared to genetic control BL10ScSnJ mice (wild-type. In vivo lung function studies using a small animal ventilator revealed a significant reduction in peak airway resistance induced by maximum concentrations of inhaled MCh in mdx mice, while there was no change in other lung function parameters. These data show that the lack of dystrophin is associated with a concomitant suppression of ASM cell phenotype maturation in vitro, ASM contraction ex vivo and lung function in vivo, indicating that a linkage between the DGC and the actin cytoskeleton via dystrophin is a determinant of the phenotype and functional properties of ASM.

  10. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Directory of Open Access Journals (Sweden)

    Rafael Rodríguez-Muñoz

    Full Text Available The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f, during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV. By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60% and multipolar Glutamatergic (≤40% neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC: dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively, in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  11. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  12. MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy

    Science.gov (United States)

    Pilotto, Sara; Gkountakos, Anastasios; Carbognin, Luisa; Scarpa, Aldo; Tortora, Giampaolo

    2017-01-01

    The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients’ subgroup, are explored. PMID:28164087

  13. MET exon 14 juxtamembrane splicing mutations: clinical and therapeutical perspectives for cancer therapy.

    Science.gov (United States)

    Pilotto, Sara; Gkountakos, Anastasios; Carbognin, Luisa; Scarpa, Aldo; Tortora, Giampaolo; Bria, Emilio

    2017-01-01

    The MET proto-oncogene plays crucial roles in cell growth and proliferation, survival and apoptosis, epithelial-mesenchymal transition (EMT) and invasion, potentially conditioning the development and progression of the carcinogenesis process. The MET-associated aberrant signaling could be triggered by a variety of mechanisms, such as mutations, gene amplification, increased gene copy number and Met/HGF protein expression. Among the various MET alterations, MET exon 14 splicing abnormalities, causing the loss of the Met juxtamembrane (JM) domain, recently emerged as a new potential oncogenic driver and have been identified and validated across different cancer and histology subtypes. Moreover, this aberration was found to be mutually exclusive with other recognized drivers, thus strongly nominating its potential oncogenic role. Recently, the clinical activity of anti-Met-targeted therapy was demonstrated particularly in patients harboring MET exon 14 skipping lung cancer, resulting in a renewed enthusiasm to further test MET precision therapy in prospective trials. In this review, the key preclinical and clinical data regarding MET exon 14 skipping splicing variants as an actionable genomic aberration in cancer are described, and the perspectives deriving from the validation of such alteration as a potential target, which may further allow driving the therapeutic approach in this molecularly selected patients' subgroup, are explored.

  14. Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

    Directory of Open Access Journals (Sweden)

    Jill A. Dembowski

    2012-01-01

    Full Text Available Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5 splice site and negative regulation by several splicing factors, including SC35 (SRSF2 and ASF/SF2 (SRSF1, drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide.

  15. Maternal and best friends' influences on meal-skipping behaviours.

    Science.gov (United States)

    Pearson, Natalie; Williams, Lauren; Crawford, David; Ball, Kylie

    2012-09-01

    Skipping meals is particularly common during adolescence and can have a detrimental effect on multiple aspects of adolescent health. Understanding the correlates of meal-skipping behaviours is important for the design of nutrition interventions. The present study examined maternal and best friends' influences on adolescent meal-skipping behaviours. Frequency of skipping breakfast, lunch and dinner was assessed using a Web-based survey completed by 3001 adolescent boys and girls from years 7 and 9 of secondary schools in Victoria, Australia. Perceived best friend and maternal meal skipping, modelling of healthy eating (eating healthy food, limiting junk food, eating fruit and vegetables) and weight watching were assessed. Best friend and maternal factors were differentially associated with meal-skipping behaviours. For example, boys and girls who perceived that their best friend often skipped meals were more likely to skip lunch (OR = 2·01, 95 % CI 1·33, 3·04 and OR = 1·93, 95 % CI 1·41, 2·65; P adolescents on how to assess and interpret unhealthy eating behaviours that they observe from significant others may be one nutrition promotion strategy to reduce meal-skipping behaviour. The involvement of mothers may be particularly important in such efforts. Encouraging a peer subculture that promotes regular consumption of meals and educates adolescents on the detrimental impact of meal-skipping behaviour on health may also offer a promising nutrition promotion strategy.

  16. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset

    Directory of Open Access Journals (Sweden)

    Nayereh Nouri

    2014-01-01

    Full Text Available Background: The Duchenne muscular dystrophy (DMD gene is located in the short arm of the X chromosome (Xp21. It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the Dystrophin gene result in DMD and Becker muscular dystrophy. In this study, the efficiency of multiplex ligation-dependent probe amplification (MLPA over multiplex polymerase chain reaction (PCR assays in an Iranian population was investigated. Materials and Methods: Multiplex PCR assays and MLPA analysis were carried out in 74 patients affected with DMD. Results: Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions. Conclusion: Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

  17. IVs to Skip for Immunizing WEP against FMS Attack

    Science.gov (United States)

    Kobara, Kazukuni; Imai, Hideki

    The WEP (Wired Equivalent Privacy) is a part of IEEE 802.11 standard designed for protecting over-the-air communication. While almost all of the WLAN (Wireless LAN) cards and the APs (Access Points) support WEP, a serious key recovery attack (aka FMS attack) was identified by Fluhrer et al. The FMS attack can basically be prevented by skipping IVs (Initial Values) used in the attack, but naive skip methods reveal information on the WEP key since most of them depend on the WEP key and the patterns of the skipped IV reveal it. In order to skip IVs safely, the skip patterns must be chosen carefully. In this paper, we review the attack conditions (6) and (7), whose success probability is the highest, 0.05, amongst all known conditions to guess one key-byte from one packet. Then we identify their safe skip patterns.

  18. Proteins, exons and molecular evolution.

    Science.gov (United States)

    Holland, S K; Blake, C C

    1987-01-01

    The discovery of the eukaryotic gene structure has prompted research into the potential relationship between protein structure and function and the corresponding exon/intron patterns. The exon shuffling hypothesis put forward by Gilbert and Blake suggests the encodement of structural and functional protein elements by exons which can recombine to create novel proteins. This provides an explanation for the relatively rapid evolution of proteins from a few primordial molecules. As the number of gene and protein structures increases, evidence of exon shuffling is becoming more apparent and examples are presented both from modern multi-domain proteins and ancient proteins. Recent work into the chemical properties and catalytic functions of RNA have led to hypotheses based upon the early existence of RNA. These theories suggest that the split gene structure originated in the primordial soup as a result of random RNA synthesis. Stable regions of RNA, or exons, were utilised as primitive enzymes. In response to selective pressures for information storage, the activity was directly transferred from the RNA enzymes or ribozymes, to proteins. These short polypeptides fused together to create larger proteins with a wide range of functions. Recent research into RNA processing and exon size, discussed in this review, provides a clearer insight into the evolutionary development of the gene and protein structure.

  19. SkipCor: skip-mention coreference resolution using linear-chain conditional random fields.

    Directory of Open Access Journals (Sweden)

    Slavko Žitnik

    Full Text Available Coreference resolution tries to identify all expressions (called mentions in observed text that refer to the same entity. Beside entity extraction and relation extraction, it represents one of the three complementary tasks in Information Extraction. In this paper we describe a novel coreference resolution system SkipCor that reformulates the problem as a sequence labeling task. None of the existing supervised, unsupervised, pairwise or sequence-based models are similar to our approach, which only uses linear-chain conditional random fields and supports high scalability with fast model training and inference, and a straightforward parallelization. We evaluate the proposed system against the ACE 2004, CoNLL 2012 and SemEval 2010 benchmark datasets. SkipCor clearly outperforms two baseline systems that detect coreferentiality using the same features as SkipCor. The obtained results are at least comparable to the current state-of-the-art in coreference resolution.

  20. Optimization Research of Mine Skip Quantitative Loading System

    Science.gov (United States)

    Wang, Shuang; Hu, Kun; Cheng, Gang; Li, De-yong

    2016-06-01

    The size and change of the impact load of coal material applied to the skip are studied aiming at the quantitative loading system of the skip. Based on the impulse theorem and with reasonable assumption, the calculation formula for impact force of the coal material is deducted and the impact characteristic of the impact force to the quantitative loading system of the skip is analyzed. The process of the coal material falling from quantitative conveyor to skip is analyzed with the discrete element simulation so that the distributed load of the impact force of the coal material at the skip bottom is obtained. The results show that the coal material produces large impact force (687 N) to the skip bottom the moment the coal material falls into the skip, and then the force decreases rapidly to about 245 N and increases gradually during the fluctuation; the impact force applied to the skip bottom increases with the increase of the coal transportation speed and the size of discharging port of the chute, but it is not in direct proportional relationship. The simulation results are basically the same as the experimental results. Finally the optimization parameters of the speed of quantitative conveyor and the size of the discharging port of the chute are searched for so as to improve the capacity of the conveyor and impact load assumed by the skip bottom.

  1. Skipped spawning in fishes: More common than you might think

    DEFF Research Database (Denmark)

    Rideout, Rick M.; Tomkiewicz, Jonna

    2011-01-01

    on elemental and isotope signatures. Skipped spawning is most commonly attributed to deficient diet and poor nutritional condition. Advances made in this field of study in recent years include descriptions of hormonal changes that precede and perhaps initiate skipped spawning, the development of life history...... research on skipped spawning, covering a broad range of fishes with diverse life history strategies. Evidence for skipped spawning has been collected by use of traditional histological techniques as well as modern technological advances, such as satellite tags and the ability to track fish movements based...

  2. Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy)

    DEFF Research Database (Denmark)

    Vorwerk, P; Christoffersen, C T; Müller, J

    1999-01-01

    domain. In the correct spliced variant, the point mutation is silent and results in a normally translated IR. The paternal allele carries a missense mutation in the tyrosine kinase domain. All three cDNA variants were present in the lymphocytes of the patients. Purified IR from 293 cells overexpressing...... to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase...... either of the two mutated receptors lacked basal or stimulated IR beta-subunit autophosphorylation. A third brother who inherited both normal alleles has an normal muscle phenotype and insulin sensitivity, suggesting a direct linkage of these IR mutations with the CFTDM phenotype....

  3. Platelet adhesion: structural and functional diversity of short dystrophin and utrophins in the formation of dystrophin-associated-protein complexes related to actin dynamics.

    Science.gov (United States)

    Cerecedo, Doris; Martínez-Rojas, Dalila; Chávez, Oscar; Martínez-Pérez, Francisco; García-Sierra, Francisco; Rendon, Alvaro; Mornet, Dominique; Mondragón, Ricardo

    2005-12-01

    Platelets are dynamic cell fragments that modify their shape during activation. Utrophin and dystrophins are minor actin-binding proteins present in muscle and non-muscle cytoskeleton. In the present study, we characterised the pattern of Dp71 isoforms and utrophin gene products by immunoblot in human platelets. Two new dystrophin isoforms were found, Dp71f and Dp71 d, as well as the Up71 isoform and the dystrophin-associated proteins, alpha and beta -dystrobrevins. Distribution of Dp71d/Dp71delta110m, Up400/Up71 and dystrophin-associated proteins in relation to the actin cytoskeleton was evaluated by confocal microscopy in both resting and platelets adhered on glass. Formation of two dystrophin-associated protein complexes (Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC) was demonstrated by co-immunoprecipitation and their distribution in relation to the actin cytoskeleton was characterised during platelet adhesion. The Dp71d/Dp71delta100m approximately DAPC is maintained mainly at the granulomere and is associated with dynamic structures during activation by adhesion to thrombin-coated surfaces. Participation of both Dp71d/Dp71delta110m approximately DAPC and Up400/Up71 approximately DAPC in the biological roles of the platelets is discussed.

  4. Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

    Science.gov (United States)

    Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

    2015-11-01

    The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.

  5. Multiplex ligation-dependent probe amplification for rapid detection of deletions and duplications in the dystrophin gene

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by mutations in the dystrophin gene. The majority of recognized mutations are copy number changes of individual exons. The objective of the present study was to assess the multiplex ligation-dependent probe amplification (MLPA) effects of detection of gene mutations. Methods: Samples of 20 control males and 80 males and their mothers referred to our diagnostic facility on the clinical suspicion of DMD or BMD were tested by MLPA and multiplex PCR. Results: The mean DQs for all peak of 20 control male samples was 1.02 (range from 0.83 to 1.21) by MLPA. Deletions or duplications were identified in 6 out of 31 families that had been previously tested as negative by multiplex PCR. One case of complex rearrangement involving a duplication of two regions: dupEX3-9 and dupEX 17-41 were found by MLPA. Conclusions: MLPA is a highly sensitive method and rapid alternative to multiplex PCR for detection of DMD and BMD.

  6. Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice

    Directory of Open Access Journals (Sweden)

    Han Renzhi

    2011-12-01

    Full Text Available Abstract Background Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with β-dystroglycan. This link extends to the extracellular matrix by β-dystroglycan's interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle. Methods To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. Results The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin

  7. Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles.

    Science.gov (United States)

    Pichavant, Christophe; Chapdelaine, Pierre; Cerri, Daniel G; Bizario, Joao C S; Tremblay, Jacques P

    2010-11-01

    Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by the absence of dystrophin (427 kDa). An approach to eventually restore this protein in patients with DMD is to introduce into their muscles a plasmid encoding dystrophin cDNA. Because the phenotype of the dystrophic dog is closer to the human phenotype than is the mdx mouse phenotype, we have studied the electrotransfer of a plasmid carrying the full-length dog dystrophin (FLDYS(dog)) in dystrophic dog muscle. To achieve this nonviral delivery, the FLDYS(dog) cDNA was cloned in two plasmids containing either a cytomegalovirus or a muscle creatine kinase promoter. In both cases, our results showed that the electrotransfer of these large plasmids (∼17 kb) into mouse muscle allowed FLDYS(dog) expression in the treated muscle. The electrotransfer of pCMV.FLDYS(dog) in a dystrophic dog muscle also led to the expression of dystrophin. In conclusion, introduction of the full-length dog dystrophin cDNA by electrotransfer into dystrophic dog muscle is a potential approach to restore dystrophin in patients with DMD. However, the electrotransfer procedure should be improved before applying it to humans.

  8. Dystrophin hydrophobic regions in the pathogenesis of Duchenne and Becker muscular dystrophies

    Directory of Open Access Journals (Sweden)

    Yingyin Liang

    2015-05-01

    Full Text Available The aim of our study was to determine the role of dystrophin hydrophobic regions in the pathogenesis of Duchenne (DMD and Becker (BMD muscular dystrophies, by the Kyte-Doolittle scale mean hydrophobicity profile and 3D molecular models. A total of 1038 cases diagnosed with DMD or BMD with the in-frame mutation were collected in our hospital and the Leiden DMD information database in the period 2002-2013. Correlation between clinical types and genotypes were determined on the basis of these two sources. In addition, the Kyte-Doolittle scale mean hydrophobicity of dystrophin was analyzed using BioEdit software and the models of the hydrophobic domains of dystrophin were constructed. The presence of four hydrophobic regions is confirmed. They include the calponin homology CH2 domain on the actin-binding domain (ABD, spectrin-type repeat 16, hinge III and the EF Hand domain. The severe symptoms of DMD usually develop as a result of the mutational disruption in the hydrophobic regions I, II and IV of dystrophin – those that bind associated proteins of the dystrophin-glycoprotein complex (DGC. On the other hand, when the hydrophobic region III is deleted, the connection of the ordered repeat domains of the central rod domain remains intact, resulting in the less severe clinical presentation. We conclude that mutational changes in the structure of hydrophobic regions of dystrophin play an important role in the pathogenesis of DMD.

  9. Comparative Training Responses to Rope Skipping and Jogging.

    Science.gov (United States)

    Buyze, Michael T.; And Others

    1986-01-01

    This study compared physiological adaptations of 26 sedentary volunteers to six-week programs of jogging and rope skipping in order to test whether 10 minutes of rope skipping is equal to 30 minutes of jogging for improved cardiovascular efficiency. Results are discussed. (Author/MT)

  10. Computational study of the human dystrophin repeats: interaction properties and molecular dynamics.

    Science.gov (United States)

    Legrand, Baptiste; Giudice, Emmanuel; Nicolas, Aurélie; Delalande, Olivier; Le Rumeur, Elisabeth

    2011-01-01

    Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD). It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.

  11. Computational study of the human dystrophin repeats: interaction properties and molecular dynamics.

    Directory of Open Access Journals (Sweden)

    Baptiste Legrand

    Full Text Available Dystrophin is a large protein involved in the rare genetic disease Duchenne muscular dystrophy (DMD. It functions as a mechanical linker between the cytoskeleton and the sarcolemma, and is able to resist shear stresses during muscle activity. In all, 75% of the dystrophin molecule consists of a large central rod domain made up of 24 repeat units that share high structural homology with spectrin-like repeats. However, in the absence of any high-resolution structure of these repeats, the molecular basis of dystrophin central domain's functions has not yet been deciphered. In this context, we have performed a computational study of the whole dystrophin central rod domain based on the rational homology modeling of successive and overlapping tandem repeats and the analysis of their surface properties. Each tandem repeat has very specific surface properties that make it unique. However, the repeats share enough electrostatic-surface similarities to be grouped into four separate clusters. Molecular dynamics simulations of four representative tandem repeats reveal specific flexibility or bending properties depending on the repeat sequence. We thus suggest that the dystrophin central rod domain is constituted of seven biologically relevant sub-domains. Our results provide evidence for the role of the dystrophin central rod domain as a scaffold platform with a wide range of surface features and biophysical properties allowing it to interact with its various known partners such as proteins and membrane lipids. This new integrative view is strongly supported by the previous experimental works that investigated the isolated domains and the observed heterogeneity of the severity of dystrophin related pathologies, especially Becker muscular dystrophy.

  12. Loss of dystrophin is associated with increased myocardial stiffness in a model of left ventricular hypertrophy.

    Science.gov (United States)

    Donato, Martín; Buchholz, Bruno; Morales, Celina; Valdez, Laura; Zaobornyj, Tamara; Baratta, Sergio; Paez, Diamela T; Matoso, Mirian; Vaccarino, Guillermo; Chejtman, Demian; Agüero, Oscar; Telayna, Juan; Navia, José; Hita, Alejandro; Boveris, Alberto; Gelpi, Ricardo J

    2017-08-01

    Transition from compensated to decompensated left ventricular hypertrophy (LVH) is accompanied by functional and structural changes. Here, the aim was to evaluate dystrophin expression in murine models and human subjects with LVH by transverse aortic constriction (TAC) and aortic stenosis (AS), respectively. We determined whether doxycycline (Doxy) prevented dystrophin expression and myocardial stiffness in mice. Additionally, ventricular function recovery was evaluated in patients 1 year after surgery. Mice were subjected to TAC and monitored for 3 weeks. A second group received Doxy treatment after TAC. Patients with AS were stratified by normal left ventricular end-diastolic wall stress (LVEDWS) and high LVEDWS, and groups were compared. In mice, LVH decreased inotropism and increased myocardial stiffness associated with a dystrophin breakdown and a decreased mitochondrial O2 uptake (MitoMVO2). These alterations were attenuated by Doxy. Patients with high LVEDWS showed similar results to those observed in mice. A correlation between dystrophin and myocardial stiffness was observed in both mice and humans. Systolic function at 1 year post-surgery was only recovered in the normal-LVEDWS group. In summary, mice and humans present diastolic dysfunction associated with dystrophin degradation. The recovery of ventricular function was observed only in patients with normal LVEDWS and without dystrophin degradation. In mice, Doxy improved MitoMVO2. Based on our results it is concluded that the LVH with high LVEDWS is associated to a degradation of dystrophin and increase of myocardial stiffness. At least in a murine model these alterations were attenuated after the administration of a matrix metalloprotease inhibitor.

  13. Efficient carry skip Adder design using full adder and carry skip block based on reversible Logic

    Directory of Open Access Journals (Sweden)

    Varun Pratap Singh

    2015-12-01

    Full Text Available In recent years, Reversible Logic is becoming more and more prominent technology having its applications in Quantum Computing, Nanotechnology, and Optical Computing. Reversibility plays an important role when energy efficient computations are considered. In this paper, binary full Adder with Design I and Design II are proposed. The performance analysis is verified using number of reversible gates, Garbage input/outputs, delay, number of logical calculations and Quantum Cost. According to the suitability of full adder design I and design II carry skip adder block is also constructed with some improvement in terms of delay in block carry generation. It is observed that Reversible carry skip Binary Adder with Design II is efficient compared to Design I

  14. Update History of This Database - SKIP Stemcell Database | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us SKIP Stemcell Database Update History of This Database Date Update contents 2017/03/13 SKIP Stemcell Database... English archive site is opened. 2013/03/29 SKIP Stemcell Database ( https://www.skip.med.k...eio.ac.jp/SKIPSearch/top?lang=en ) is opened. About This Database Database Description Download License Upda...te History of This Database Site Policy | Contact Us Update History of This Database - SKIP Stemcell Database | LSDB Archive ...

  15. PRO-051, an antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy.

    Science.gov (United States)

    Hammond, Suzan M; Wood, Matthew Ja

    2010-08-01

    PRO-051 (GSK-2402968), being developed by GlaxoSmithKline plc, under license from Leiden University Medical Center and Prosensa Therapeutics BV, is a 2'-O-methyl phosphorothioate antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy (DMD). The PRO-051 oligonucleotide sequence induces skipping of exon 51 of the dystrophin gene by binding to a sequence within the dystrophin pre-mRNA and masking the exon inclusion signals that are used for splicing. Removal of exon 51 from an exon 45 to 50, 47 to 50, 48 to 50, 49 to 50, 50, 52 or 52 to 63 deleted transcript allows restoration of the open reading frame and synthesis of an internally truncated, semi-functional dystrophin protein. By targeting exon 51, approximately 13% of patients with DMD could be treated, the largest proportion of patients that could benefit from targeting a single dystrophin exon. A proof-of-concept clinical trial of PRO-051 in patients with DMD demonstrated that a single intramuscular administration of PRO-051 induced exon skipping within muscle fibers adjacent to the injection site, while biopsies revealed dystrophin expression in treated but not control muscle fibers. At the time of publication, a phase I/IIa trial to evaluate subcutaneous delivery of PRO-051 had been completed, although full results were yet to be published.

  16. Kinematic characteristics of motor patterns in rope skipping

    OpenAIRE

    Luiz Henrique da Silva; Ana Maria Pellegrini

    2009-01-01

    Rope skipping seems to be an easy task to be performed. However, careful analysis of this motor skill shows how complex the execution of this task is. The objective of this study was to examine kinematic variables of jump patterns as a function of skipping frequency. Eight male university students performed a sequence of 30 rope jumps using two jump patterns (alternating support of the feet and simultaneous support of the feet) at three skipping frequencies (1.5, 1.7,1.9 Hz). Frequencies were...

  17. Preservation of Muscle Force in Mdx3cv Mice Correlates with Low-Level Expression of a Near Full-Length Dystrophin Protein

    OpenAIRE

    2008-01-01

    The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx...

  18. Ex vivo gene editing of the dystrophin gene in muscle stem cells mediated by peptide nucleic acid single stranded oligodeoxynucleotides induces stable expression of dystrophin in a mouse model for Duchenne muscular dystrophy.

    Science.gov (United States)

    Nik-Ahd, Farnoosh; Bertoni, Carmen

    2014-07-01

    Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutations in the dystrophin gene, which result in the complete absence of dystrophin protein throughout the body. Gene correction strategies hold promise to treating DMD. Our laboratory has previously demonstrated the ability of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to permanently correct single-point mutations at the genomic level. In this study, we show that PNA-ssODNs can target and correct muscle satellite cells (SCs), a population of stem cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SCs transfected with correcting PNA-ssODNs were able to engraft and to restore dystrophin expression. The number of dystrophin-positive fibers was shown to significantly increase over time. Expression was confirmed to be the result of the activation of a subpopulation of SCs that had undergone repair as demonstrated by immunofluorescence analyses of engrafted muscles using antibodies specific to full-length dystrophin transcripts and by genomic DNA analysis of dystrophin-positive fibers. Furthermore, the increase in dystrophin expression detected over time resulted in a significant improvement in muscle morphology. The ability of transplanted cells to return into quiescence and to activate upon demand was confirmed in all engrafted muscles following injury. These results demonstrate the feasibility of using gene editing strategies to target and correct SCs and further establish the therapeutic potential of this approach to permanently restore dystrophin expression into muscle of DMD patients.

  19. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division.

    Science.gov (United States)

    Dumont, Nicolas A; Wang, Yu Xin; von Maltzahn, Julia; Pasut, Alessandra; Bentzinger, C Florian; Brun, Caroline E; Rudnicki, Michael A

    2015-12-01

    Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.

  20. Parental source effect of inherited mutations in the dystrophin gene of mice and men

    Energy Technology Data Exchange (ETDEWEB)

    Kress, W.; Grimm, T.; Mueller, C.R. [Institute of Human Genetics, Wuerburg (Germany); Bittner, R. [Institute of Anatomy, Wein (Australia)

    1994-09-01

    Skewed X-inactivation has been suspected the genetic cause for some manifesting female carriers of BMD and DMD. To test whether a parental source effect on the protein expression of the dystrophin gene exists, we have set up backcrosses of mdx mice to wild type strains, enabling us to study the effect of the well-defined origin of the mutation on the dystrophin expression. In skeletal muscle sections the immunohistological staining patterns of dystrophin antibodies were showing a significant difference in the proportion of dystrophin positive versus negative fibers, suggesting a lower expression of paternally inherited mdx mutations. These data are in concordance with the pyruvate kinase (PK) levels in the serum: PK levels were much higher when the mutation was of maternal origin as compared to PK levels in paternally derived mutations. In order to test this {open_quotes}paternal source effect{close_quotes} in humans, we checked obligatory carriers of Becker muscular dystrophy (BMD) for the origin of their mutations. Creatin kinase (CK) levels in 21 carriers with maternally derived mutations were compared to CK values from 8 heterozygotes with mutations of paternal origin: CK (mat) = 140.3 IU/1 versus CK (pat) = 48.6 IU/I. The difference is statistically significant at the 5% level. These observations suggest either a differential X-inactivation or an imprinting of the dystrophin gene in mice and men.

  1. Deficiency in Cardiac Dystrophin Affects the Abundance of the α-/β-Dystroglycan Complex

    Directory of Open Access Journals (Sweden)

    James Lohan

    2005-01-01

    Full Text Available Although Duchenne muscular dystrophy is primarily categorised as a skeletal muscle disease, deficiency in the membrane cytoskeletal protein dystrophin also affects the heart. The central transsarcolemmal linker between the actin membrane cytoskeleton and the extracellular matrix is represented by the dystrophin-associated dystroglycans. Chemical cross-linking analysis revealed no significant differences in the dimeric status of the α-/β-dystroglycan subcomplex in the dystrophic mdx heart as compared to normal cardiac tissue. In analogy to skeletal muscle fibres, heart muscle also exhibited a greatly reduced abundance of both dystroglycans in dystrophin-deficient cells. Immunoblotting demonstrated that the degree of reduction in α-dystroglycan is more pronounced in matured mdx skeletal muscle as contrasted to the mdx heart. The fact that the deficiency in dystrophin triggers a similar pathobiochemical response in both types of muscle suggests that the cardiomyopathic complications observed in x-linked muscular dystrophy might be initiated by the loss of the dystrophin-associated surface glycoprotein complex.

  2. Modeling and Analysis of Pulse Skip Modulation

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The state space average model and the large signal models of Pulse Skip Modulation (PSM) mode are given in this paper. Farther more, based on these models and simulations of PSM converter circuits, the analysis of the characteristics of PSM converter is described in this paper, of which include efficiency, frequency spectrum analysis, output voltage ripple, response speed and interference rejection capability. Compared with PWM control mode, PSM converter has high efficiency, especially with light loads, quick response, good interference rejection and good EMC characteristic. Improved PSM slightly, it could be a kind of good independent regulating mode during the whole operating process for a DC-DC converter. Finally, some experimental results are also presented in this paper.

  3. GAA Deficiency in Pompe Disease Is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells

    Directory of Open Access Journals (Sweden)

    Erik van der Wal

    2017-06-01

    Full Text Available Pompe disease is a metabolic myopathy caused by deficiency of the acid α-glucosidase (GAA enzyme and results in progressive wasting of skeletal muscle cells. The c.-32-13T>G (IVS1 GAA variant promotes exon 2 skipping during pre-mRNA splicing and is the most common variant for the childhood/adult disease form. We previously identified antisense oligonucleotides (AONs that promoted GAA exon 2 inclusion in patient-derived fibroblasts. It was unknown how these AONs would affect GAA splicing in skeletal muscle cells. To test this, we expanded induced pluripotent stem cell (iPSC-derived myogenic progenitors and differentiated these to multinucleated myotubes. AONs restored splicing in myotubes to a similar extent as in fibroblasts, suggesting that they act by modulating the action of shared splicing regulators. AONs targeted the putative polypyrimidine tract of a cryptic splice acceptor site that was part of a pseudo exon in GAA intron 1. Blocking of the cryptic splice donor of the pseudo exon with AONs likewise promoted GAA exon 2 inclusion. The simultaneous blocking of the cryptic acceptor and cryptic donor sites restored the majority of canonical splicing and alleviated GAA enzyme deficiency. These results highlight the relevance of cryptic splicing in human disease and its potential as therapeutic target for splicing modulation using AONs.

  4. Skip the Antibiotics for Mild Eczema in Kids

    Science.gov (United States)

    ... medlineplus.gov/news/fullstory_164083.html Skip the Antibiotics for Mild Eczema in Kids Skin condition cleared ... March 14, 2017 (HealthDay News) -- Despite widespread use, antibiotics are not an effective treatment for milder cases ...

  5. Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes.

    Science.gov (United States)

    Rubi, Lena; Koenig, Xaver; Kubista, Helmut; Todt, Hannes; Hilber, Karlheinz

    2017-03-04

    Kir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current IK1, which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential IK1 abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that IK1 was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.

  6. The sarcoglycan-sarcospan complex localization in mouse retina is independent from dystrophins

    Science.gov (United States)

    Fort, Patrice; Estrada, Francisco-Javier; Bordais, Agnès; Mornet, Dominique; Sahel, José-Alain; Picaud, Serge; Vargas, Haydeé Rosas; Coral-Vázquez, Ramón M.; Rendon, Alvaro

    2005-01-01

    The sarcoglycan–sarcospan (SG–SSPN) complex is part of the dystrophin-glycoprotein complex that has been extensively characterized in muscle. To establish the framework for functional studies of sarcoglycans in retina here, we quantified sarcoglycans mRNA levels with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and performed immunohistochemistry to determine their cellular and subcellular distribution. We showed that the β-, δ-, γ-, ε-sarcoglycans and sarcospan are expressed in mouse retina. They are localized predominantly in the outer and the inner limiting membranes, probably in the Müller cells and also in the ganglion cells axons where the expression of dystrophins have never been reported. We also investigated the status of the sarcoglycans in the retina of mdx3cv mutant mice for all Duchene Muscular Dystrophy (DMD) gene products. The absence of dystrophin did not produce any change in the sarcoglycan–sarcospan components expression and distribution. PMID:15993965

  7. Snacking behaviours of adolescents and their association with skipping meals

    OpenAIRE

    Worsley Anthony; Ball Kylie; MacFarlane Abbie; Savige Gayle; Crawford David

    2007-01-01

    Abstract Background Snacking is likely to play an important role in the development of overweight and obesity, yet little is known about the contexts of snacking in adolescents or how snacking may influence other dietary habits, like meal skipping. This study examines the contexts in which adolescents snack and whether these contexts are associated with demographic characteristics of adolescents and with meal skipping. Methods A cross-sectional, self-reported online food habits survey was adm...

  8. Optimal design method for fast carry-skip adders

    Science.gov (United States)

    Lee, Songjun; Swartzlander, Earl E., Jr.

    2001-11-01

    A carry-skip adder is faster than a ripple carry adder and it has a simple structure. To maximize the speed it is necessary to optimize the width of the blocks that comprise the carry skip adder. This paper presents a simple algorithm to select the size of each block. Assuming that each logic gate has a unit delay, the algorithm achieves slightly faster designs for 64 and 128 bit adders than previous methods developed by Guyot, et al. and Kantabutra.

  9. Quantization Skipping Method for H.264/AVC Video Coding

    Institute of Scientific and Technical Information of China (English)

    Won-seon SONG; Min-cheol HONG

    2010-01-01

    This paper presents a quantization skipping method for H.264/AVC video coding standard. In order to reduce the computational-cost of quantization process coming from integer discrete cosine transform of H.264/AVC, a quantization skipping condition is derived by the analysis of integer transform and quantization procedures. The experimental results show that the proposed algorithm has the capability to reduce the computational cost about 10%~25%.

  10. Regional genomic instability predisposes to complex dystrophin gene rearrangements.

    Science.gov (United States)

    Oshima, Junko; Magner, Daniel B; Lee, Jennifer A; Breman, Amy M; Schmitt, Eric S; White, Lisa D; Crowe, Carol A; Merrill, Michelle; Jayakar, Parul; Rajadhyaksha, Aparna; Eng, Christine M; del Gaudio, Daniela

    2009-09-01

    Mutations in the dystrophin gene (DMD) cause Duchenne and Becker muscular dystrophies and the majority of cases are due to DMD gene rearrangements. Despite the high incidence of these aberrations, little is known about their causative molecular mechanism(s). We examined 792 DMD/BMD clinical samples by oligonucleotide array-CGH and report on the junction sequence analysis of 15 unique deletion cases and three complex intragenic rearrangements to elucidate potential underlying mechanism(s). Furthermore, we present three cases with intergenic rearrangements involving DMD and neighboring loci. The cases with intragenic rearrangements include an inversion with flanking deleted sequences; a duplicated segment inserted in direct orientation into a deleted region; and a splicing mutation adjacent to a deletion. Bioinformatic analysis demonstrated that 7 of 12 breakpoints combined among 3 complex cases aligned with repetitive sequences, as compared to 4 of 30 breakpoints for the 15 deletion cases. Moreover, the inversion/deletion case may involve a stem-loop structure that has contributed to the initiation of this rearrangement. For the duplication/deletion and splicing mutation/deletion cases, the presence of the first mutation, either a duplication or point mutation, may have elicited the deletion events in an attempt to correct preexisting mutations. While NHEJ is one potential mechanism for these complex rearrangements, the highly complex junction sequence of the inversion/deletion case suggests the involvement of a replication-based mechanism. Our results support the notion that regional genomic instability, aided by the presence of repetitive elements, a stem-loop structure, and possibly preexisting mutations, may elicit complex rearrangements of the DMD gene.

  11. The emergence of alternative 3' and 5' splice site exons from constitutive exons.

    Directory of Open Access Journals (Sweden)

    Eli Koren

    2007-05-01

    Full Text Available Alternative 3' and 5' splice site (ss events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3'ss and 5'ss exons. The results revealed that alternative 3'ss and 5'ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3'ss and 5'ss exons exhibit low levels of symmetry (frame-preserving, similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.

  12. Efficient and fast functional screening of microdystrophin constructs in vivo and in vitro for therapy of duchenne muscular dystrophy

    DEFF Research Database (Denmark)

    Jørgensen, Louise Helskov; Larochelle, Nancy; Orlopp, Kristian

    2009-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked, lethal genetic disorder affecting the skeletal muscle compartment, and is caused by mutation(s) in the dystrophin gene. Gene delivery of microdystrophin constructs using adeno-associated virus (AAV) and antisense-mediated exon skipping restoring...

  13. Identification of protein features encoded by alternative exons using Exon Ontology.

    Science.gov (United States)

    Tranchevent, Léon-Charles; Aubé, Fabien; Dulaurier, Louis; Benoit-Pilven, Clara; Rey, Amandine; Poret, Arnaud; Chautard, Emilie; Mortada, Hussein; Desmet, François-Olivier; Chakrama, Fatima Zahra; Moreno-Garcia, Maira Alejandra; Goillot, Evelyne; Janczarski, Stéphane; Mortreux, Franck; Bourgeois, Cyril F; Auboeuf, Didier

    2017-06-01

    Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information. © 2017 Tranchevent et al.; Published by Cold Spring Harbor Laboratory Press.

  14. Exon circularization in mammalian nuclear extracts.

    Science.gov (United States)

    Pasman, Z; Been, M D; Garcia-Blanco, M A

    1996-06-01

    Correct ligation of exons in pre-mRNA splicing requires splice site juxtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice site, however, is predicted to result in a circular RNA. This mode of splice site pairing across the axon has been hypothesized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site pairing has been postulated to explain the formation of SRY circular transcripts in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that splice site pairing across the exon can result in exon circularization in vitro. These results indicate that spliceosome-mediated axon circularization indeed can account for the formation of scrambled exons and circular RNAs. Exon circularization efficiency decreased dramatically as the length of the exon was increased from 95 nt to 274 nt. Circularization of this longer exon was restored, however, when intronic complementary sequences were included in the RNA substrate. These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing. Therefore, the splicing of this structured RNA recapitulated SRY-like exon circularization in vitro.

  15. Dystrophin deficiency leads to disturbance of LAMP1-vesicle-associated protein secretion

    DEFF Research Database (Denmark)

    Duguez, S.; Duddy, W.; Johnston, H.

    2013-01-01

    Duchenne muscular dystrophy results from loss of the protein dystrophin, which links the intracellular cytoskeletal network with the extracellular matrix, but deficiency in this function does not fully explain the onset or progression of the disease. While some intracellular events involved...... of new potential therapeutic targets....

  16. Proteomic Profiling of the Dystrophin-Deficient mdx Phenocopy of Dystrophinopathy-Associated Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Ashling Holland

    2014-01-01

    Full Text Available Cardiorespiratory complications are frequent symptoms of Duchenne muscular dystrophy, a neuromuscular disorder caused by primary abnormalities in the dystrophin gene. Loss of cardiac dystrophin initially leads to changes in dystrophin-associated glycoproteins and subsequently triggers secondarily sarcolemmal disintegration, fibre necrosis, fibrosis, fatty tissue replacement, and interstitial inflammation. This results in progressive cardiac disease, which is the cause of death in a considerable number of patients afflicted with X-linked muscular dystrophy. In order to better define the molecular pathogenesis of this type of cardiomyopathy, several studies have applied mass spectrometry-based proteomics to determine proteome-wide alterations in dystrophinopathy-associated cardiomyopathy. Proteomic studies included both gel-based and label-free mass spectrometric surveys of dystrophin-deficient heart muscle from the established mdx animal model of dystrophinopathy. Comparative cardiac proteomics revealed novel changes in proteins associated with mitochondrial energy metabolism, glycolysis, signaling, iron binding, antibody response, fibre contraction, basal lamina stabilisation, and cytoskeletal organisation. This review summarizes the importance of studying cardiomyopathy within the field of muscular dystrophy research, outlines key features of the mdx heart and its suitability as a model system for studying cardiac pathogenesis, and discusses the impact of recent proteomic findings for exploring molecular and cellular aspects of cardiac abnormalities in inherited muscular dystrophies.

  17. The polyproline site in hinge 2 influences the functional capacity of truncated dystrophins.

    Directory of Open Access Journals (Sweden)

    Glen B Banks

    2010-05-01

    Full Text Available Mutations in dystrophin can lead to Duchenne muscular dystrophy or the more mild form of the disease, Becker muscular dystrophy. The hinge 3 region in the rod domain of dystrophin is particularly prone to deletion mutations. In-frame deletions of hinge 3 are predicted to lead to BMD, however the severity of disease can vary considerably. Here we performed extensive structure-function analyses of truncated dystrophins with modified hinges and spectrin-like repeats in mdx mice. We found that the polyproline site in hinge 2 profoundly influences the functional capacity of a microdystrophin(DeltaR4-R23/DeltaCT with a large deletion in the hinge 3 region. Inclusion of polyproline in microdystrophin(DeltaR4-R23/DeltaCT led to small myofibers (12% smaller than wild-type, Achilles myotendinous disruption, ringed fibers, and aberrant neuromuscular junctions in the mdx gastrocnemius muscles. Replacing hinge 2 of microdystrophin(DeltaR4-R23/DeltaCT with hinge 3 significantly improved the functional capacity to prevent muscle degeneration, increase muscle fiber area, and maintain the junctions. We conclude that the rigid alpha-helical structure of the polyproline site significantly impairs the functional capacity of truncated dystrophins to maintain appropriate connections between the cytoskeleton and extracellular matrix.

  18. Studying the role of dystrophin-associated proteins in influencing Becker muscular dystrophy disease severity.

    Science.gov (United States)

    van den Bergen, J C; Wokke, B H A; Hulsker, M A; Verschuuren, J J G M; Aartsma-Rus, A M

    2015-03-01

    Becker muscular dystrophy is characterized by a variable disease course. Many factors have been implicated to contribute to this diversity, among which the expression of several components of the dystrophin associated glycoprotein complex. Together with dystrophin, most of these proteins anchor the muscle fiber cytoskeleton to the extracellular matrix, thus protecting the muscle from contraction induced injury, while nNOS is primarily involved in inducing vasodilation during muscle contraction, enabling adequate muscle oxygenation. In the current study, we investigated the role of three components of the dystrophin associated glycoprotein complex (beta-dystroglycan, gamma-sarcoglycan and nNOS) and the dystrophin homologue utrophin on disease severity in Becker patients. Strength measurements, data about disease course and fresh muscle biopsies of the anterior tibial muscle were obtained from 24 Becker patients aged 19 to 66. The designation of Becker muscular dystrophy in this study was based on the mutation and not on the clinical severity. Contrary to previous studies, we were unable to find a relationship between expression of nNOS, beta-dystroglycan and gamma-sarcoglycan at the sarcolemma and disease severity, as measured by muscle strength in five muscle groups and age at reaching several disease milestones. Unexpectedly, we found an inverse correlation between utrophin expression at the sarcolemma and age at reaching disease milestones.

  19. Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

    Science.gov (United States)

    Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

    2015-01-01

    In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene.

  20. Skeletal Muscle Differentiation on a Chip Shows Human Donor Mesoangioblasts' Efficiency in Restoring Dystrophin in a Duchenne Muscular Dystrophy Model.

    Science.gov (United States)

    Serena, Elena; Zatti, Susi; Zoso, Alice; Lo Verso, Francesca; Tedesco, F Saverio; Cossu, Giulio; Elvassore, Nicola

    2016-12-01

    : Restoration of the protein dystrophin on muscle membrane is the goal of many research lines aimed at curing Duchenne muscular dystrophy (DMD). Results of ongoing preclinical and clinical trials suggest that partial restoration of dystrophin might be sufficient to significantly reduce muscle damage. Different myogenic progenitors are candidates for cell therapy of muscular dystrophies, but only satellite cells and pericytes have already entered clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from DMD patients, using a microengineered model. We designed an ad hoc experimental strategy to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. It is based on the coculture, at different ratios, of human dystrophin-positive myogenic progenitors and dystrophin-negative myoblasts in a substrate with muscle-like physiological stiffness and cell micropatterns. Results showed that both healthy myoblasts and mesoangioblasts restored dystrophin expression in DMD myotubes. However, mesoangioblasts showed unexpected efficiency with respect to myoblasts in dystrophin production in terms of the amount of protein produced (40% vs. 15%) and length of the dystrophin membrane domain (210-240 µm vs. 40-70 µm). These results show that our microscaled in vitro model of human DMD skeletal muscle validated previous in vivo preclinical work and may be used to predict efficacy of new methods aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo, reducing time, costs, and variability of clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of human mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from

  1. One Way to Design a Valence-Skip Compound

    Science.gov (United States)

    Hase, I.; Yanagisawa, T.; Kawashima, K.

    2017-02-01

    Valence-skip compound is a good candidate with high T c and low anisotropy because it has a large attractive interaction at the site of valence-skip atom. However, it is not easy to synthesize such compound because of (i) the instability of the skipping valence state, (ii) the competing charge order, and (iii) that formal valence may not be true in some compounds. In the present study, we show several examples of the valence-skip compounds and discuss how we can design them by first principles calculations. Furthermore, we calculated the electronic structure of a promising candidate of valence skipping compound RbTlCl3 from first principles. We confirmed that the charge-density wave (CDW) is formed in this compound, and the Tl atoms in two crystallographic different sites take the valence Tl1+ and Tl3+. Structure optimization study reveals that this CDW is stable at the ambient pressure, while this CDW gap can be collapsed when we apply pressure with several gigapascals. In this metallic phase, we can expect a large charge fluctuation and a large electron-phonon interaction.

  2. Skipped words and fixated words are processed differently during reading.

    Science.gov (United States)

    Eskenazi, Michael A; Folk, Jocelyn R

    2015-04-01

    The purpose of this study was to investigate whether words are processed differently when they are fixated during silent reading than when they are skipped. According to a serial processing model of eye movement control (e.g., EZ Reader) skipped words are fully processed (Reichle, Rayner, Pollatsek, Behavioral and Brain Sciences, 26(04):445-476, 2003), whereas in a parallel processing model (e.g., SWIFT) skipped words do not need to be fully processed (Engbert, Nuthmann, Richter, Kliegl, Psychological Review, 112(4):777-813, 2005). Participants read 34 sentences with target words embedded in them while their eye movements were recorded. All target words were three-letter, low-frequency, and unpredictable nouns. After the reading session, participants completed a repetition priming lexical decision task with the target words from the reading session included as the repetition prime targets, with presentation of those same words during the reading task acting as the prime. When participants skipped a word during the reading session, their reaction times on the lexical decision task were significantly longer (M = 656.42 ms) than when they fixated the word (M = 614.43 ms). This result provides evidence that skipped words are sometimes not processed to the same degree as fixated words during reading.

  3. Breakfast Skipping, Extreme Commutes, and the Sex Composition at Birth.

    Science.gov (United States)

    Mazumder, Bhashkar; Seeskin, Zachary

    2015-01-01

    A growing body of literature has shown that environmental exposures in the period around conception can affect the sex ratio at birth through selective attrition that favors the survival of female conceptuses. Glucose availability is considered a key indicator of the fetal environment, and its absence as a result of meal skipping may inhibit male survival. We hypothesize that breakfast skipping during pregnancy may lead to a reduction in the fraction of male births. Using time use data from the United States we show that women with commute times of 90 minutes or longer are 20 percentage points more likely to skip breakfast. Using U.S. census data we show that women with commute times of 90 minutes or longer are 1.2 percentage points less likely to have a male child under the age of 2. Under some assumptions, this implies that routinely skipping breakfast around the time of conception leads to a 6 percentage point reduction in the probability of a male child. Skipping breakfast during pregnancy may therefore constitute a poor environment for fetal health more generally.

  4. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.

    Science.gov (United States)

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

  5. ExonMiner: Web service for analysis of GeneChip Exon array data

    Directory of Open Access Journals (Sweden)

    Imoto Seiya

    2008-11-01

    Full Text Available Abstract Background Some splicing isoform-specific transcriptional regulations are related to disease. Therefore, detection of disease specific splice variations is the first step for finding disease specific transcriptional regulations. Affymetrix Human Exon 1.0 ST Array can measure exon-level expression profiles that are suitable to find differentially expressed exons in genome-wide scale. However, exon array produces massive datasets that are more than we can handle and analyze on personal computer. Results We have developed ExonMiner that is the first all-in-one web service for analysis of exon array data to detect transcripts that have significantly different splicing patterns in two cells, e.g. normal and cancer cells. ExonMiner can perform the following analyses: (1 data normalization, (2 statistical analysis based on two-way ANOVA, (3 finding transcripts with significantly different splice patterns, (4 efficient visualization based on heatmaps and barplots, and (5 meta-analysis to detect exon level biomarkers. We implemented ExonMiner on a supercomputer system in order to perform genome-wide analysis for more than 300,000 transcripts in exon array data, which has the potential to reveal the aberrant splice variations in cancer cells as exon level biomarkers. Conclusion ExonMiner is well suited for analysis of exon array data and does not require any installation of software except for internet browsers. What all users need to do is to access the ExonMiner URL http://ae.hgc.jp/exonminer. Users can analyze full dataset of exon array data within hours by high-level statistical analysis with sound theoretical basis that finds aberrant splice variants as biomarkers.

  6. ExonMiner: Web service for analysis of GeneChip Exon array data

    Science.gov (United States)

    Numata, Kazuyuki; Yoshida, Ryo; Nagasaki, Masao; Saito, Ayumu; Imoto, Seiya; Miyano, Satoru

    2008-01-01

    Background Some splicing isoform-specific transcriptional regulations are related to disease. Therefore, detection of disease specific splice variations is the first step for finding disease specific transcriptional regulations. Affymetrix Human Exon 1.0 ST Array can measure exon-level expression profiles that are suitable to find differentially expressed exons in genome-wide scale. However, exon array produces massive datasets that are more than we can handle and analyze on personal computer. Results We have developed ExonMiner that is the first all-in-one web service for analysis of exon array data to detect transcripts that have significantly different splicing patterns in two cells, e.g. normal and cancer cells. ExonMiner can perform the following analyses: (1) data normalization, (2) statistical analysis based on two-way ANOVA, (3) finding transcripts with significantly different splice patterns, (4) efficient visualization based on heatmaps and barplots, and (5) meta-analysis to detect exon level biomarkers. We implemented ExonMiner on a supercomputer system in order to perform genome-wide analysis for more than 300,000 transcripts in exon array data, which has the potential to reveal the aberrant splice variations in cancer cells as exon level biomarkers. Conclusion ExonMiner is well suited for analysis of exon array data and does not require any installation of software except for internet browsers. What all users need to do is to access the ExonMiner URL . Users can analyze full dataset of exon array data within hours by high-level statistical analysis with sound theoretical basis that finds aberrant splice variants as biomarkers. PMID:19036125

  7. Delay Efficient 32-Bit Carry-Skip Adder

    Directory of Open Access Journals (Sweden)

    Yu Shen Lin

    2008-01-01

    Full Text Available The design of a 32-bit carry-skip adder to achieve minimum delay is presented in this paper. A fast carry look-ahead logic using group generate and group propagate functions is used to speed up the performance of multiple stages of ripple carry adders. The group generate and group propagate functions are generated in parallel with the carry generation for each block. The optimum block sizes are decided by considering the critical path into account. The new architecture delivers the sum and carry outputs in lesser unit delays than existing carry-skip adders. The adder is implemented in 0.25 m CMOS technology at 3.3 V. The critical delay for the proposed adder is 3.4 nanoseconds. The simulation results show that the proposed adder is 18% faster than the current fastest carry-skip adder.

  8. Metabolic and Cardiorespiratory Responses of Young Women to Skipping and Jogging.

    Science.gov (United States)

    Allen, T. Earl; And Others

    1987-01-01

    Nine 18- to 29-year-old females were studied while jogging and skipping at treadmill speeds of 4.0, 4.8, and 5.4 miles per hour. Comparison of metabolic demand, musculoskeletal stress, and perceived exertion indicated skipping imposed significantly greater metabolic demands and caused higher heart rates than jogging. Skipping was also rated more…

  9. Social inequalities in young children's meal skipping behaviors: The Generation R Study

    NARCIS (Netherlands)

    A.I. Wijtzes (Anne); W. Jansen (Wilma); V.W.V. Jaddoe (Vincent W. V.); O.H. Franco (Oscar); A. Hofman (Albert); F.J. van Lenthe (Frank); H. Raat (Hein)

    2015-01-01

    textabstractBackground: Regular meal consumption is considered an important aspect of a healthy diet. While ample evidence shows social inequalities in breakfast skipping among adolescents, little is known about social inequalities in breakfast skipping and skipping of other meals among young

  10. Social inequalities in young children's meal skipping behaviors: The Generation R Study

    NARCIS (Netherlands)

    A.I. Wijtzes (Anne); W. Jansen (Wilma); V.W.V. Jaddoe (Vincent W. V.); O.H. Franco (Oscar); A. Hofman (Albert); F.J. van Lenthe (Frank); H. Raat (Hein)

    2015-01-01

    textabstractBackground: Regular meal consumption is considered an important aspect of a healthy diet. While ample evidence shows social inequalities in breakfast skipping among adolescents, little is known about social inequalities in breakfast skipping and skipping of other meals among young school

  11. Children's Perceptions of Parental Attitude Affecting Breakfast Skipping in Primary Sixth-Grade Students

    Science.gov (United States)

    Cheng, Tereza Sy; Tse, Lap Ah; Yu, Ignatius Tak-Sun; Griffiths, Sian

    2008-01-01

    Background: Breakfast skipping is an international public health concern. This study investigated the prevalence of breakfast skipping among primary sixth-grade students in Hong Kong and the impact of students' perceptions of parental attitudes on breakfast skipping. Methods: A total of 426 students aged 10-14 years in 4 local schools participated…

  12. Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

    Science.gov (United States)

    Aragón, Jorge; González-Reyes, Mayram; Romo-Yáñez, José; Vacca, Ophélie; Aguilar-González, Guadalupe; Rendón, Alvaro; Vaillend, Cyrille; Montañez, Cecilia

    2017-01-27

    Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

  13. Phase 2a study of ataluren-mediated dystrophin production in patients with nonsense mutation Duchenne muscular dystrophy

    National Research Council Canada - National Science Library

    Finkel, Richard S; Flanigan, Kevin M; Wong, Brenda; Bönnemann, Carsten; Sampson, Jacinda; Sweeney, H Lee; Reha, Allen; Northcutt, Valerie J; Elfring, Gary; Barth, Jay; Peltz, Stuart W

    2013-01-01

    Approximately 13% of boys with Duchenne muscular dystrophy (DMD) have a nonsense mutation in the dystrophin gene, resulting in a premature stop codon in the corresponding mRNA and failure to generate a functional protein. Ataluren (PTC124...

  14. Snacking behaviours of adolescents and their association with skipping meals

    Directory of Open Access Journals (Sweden)

    Worsley Anthony

    2007-09-01

    Full Text Available Abstract Background Snacking is likely to play an important role in the development of overweight and obesity, yet little is known about the contexts of snacking in adolescents or how snacking may influence other dietary habits, like meal skipping. This study examines the contexts in which adolescents snack and whether these contexts are associated with demographic characteristics of adolescents and with meal skipping. Methods A cross-sectional, self-reported online food habits survey was administered to 3,250 secondary students in years seven and nine. The students were drawn from 37 secondary schools in Victoria, Australia during 2004–2005. Frequencies of meal skipping, and snacking in eight contexts, were compared across gender, year level and region of residence. Logistic regressions were performed to examine associations between snacking contexts and meal skipping adjusting for gender and region. Results The most common contexts for snacking among adolescents were after school (4.6 times per week, while watching TV (3.5 times per week and while hanging out with friends (2.4 times per week. Adolescents were least likely to snack all day long (0.8 times per week or in the middle of the night (0.4 times per week. Snacking contexts were variously associated with gender, year level and region. In contrast, meal skipping was associated with gender and region of residence but not year level. Adolescents who reported more frequent snacking on the run, on the way to or from school, all day long, or in the middle of the night were more likely to skip meals. Conclusion These data suggest adolescents snack frequently, especially in their leisure time. In addition, adolescents who snack on the run, on the way to or from school, all day long or in the middle of the night are more likely to skip meals than are adolescents who don't snack at these times. Understanding the contexts in which adolescents snack, and their associations with skipping meals

  15. Remarkable selective constraints on exonic dinucleotide repeats.

    Science.gov (United States)

    Haasl, Ryan J; Payseur, Bret A

    2014-09-01

    Long dinucleotide repeats found in exons present a substantial mutational hazard: mutations at these loci occur often and generate frameshifts. Here, we provide clear and compelling evidence that exonic dinucleotides experience strong selective constraint. In humans, only 18 exonic dinucleotides have repeat lengths greater than six, which contrasts sharply with the genome-wide distribution of dinucleotides. We genotyped each of these dinucleotides in 200 humans from eight 1000 Genomes Project populations and found a near-absence of polymorphism. More remarkably, divergence data demonstrate that repeat lengths have been conserved across the primate phylogeny in spite of what is likely considerable mutational pressure. Coalescent simulations show that even a very low mutation rate at these loci fails to explain the anomalous patterns of polymorphism and divergence. Our data support two related selective constraints on the evolution of exonic dinucleotides: a short-term intolerance for any change to repeat length and a long-term prevention of increases to repeat length. In general, our results implicate purifying selection as the force that eliminates new, deleterious mutants at exonic dinucleotides. We briefly discuss the evolution of the longest exonic dinucleotide in the human genome--a 10 x CA repeat in fibroblast growth factor receptor-like 1 (FGFRL1)--that should possess a considerably greater mutation rate than any other exonic dinucleotide and therefore generate a large number of deleterious variants. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

  16. How are exons encoding transmembrane sequences distributed in the exon-intron structure of genes?

    Science.gov (United States)

    Sawada, Ryusuke; Mitaku, Shigeki

    2011-01-01

    The exon-intron structure of eukaryotic genes raises a question about the distribution of transmembrane regions in membrane proteins. Were exons that encode transmembrane regions formed simply by inserting introns into preexisting genes or by some kind of exon shuffling? To answer this question, the exon-per-gene distribution was analyzed for all genes in 40 eukaryotic genomes with a particular focus on exons encoding transmembrane segments. In 21 higher multicellular eukaryotes, the percentage of multi-exon genes (those containing at least one intron) within all genes in a genome was high (>70%) and with a mean of 87%. When genes were grouped by the number of exons per gene in higher eukaryotes, good exponential distributions were obtained not only for all genes but also for the exons encoding transmembrane segments, leading to a constant ratio of membrane proteins independent of the exon-per-gene number. The positional distribution of transmembrane regions in single-pass membrane proteins showed that they are generally located in the amino or carboxyl terminal regions. This nonrandom distribution of transmembrane regions explains the constant ratio of membrane proteins to the exon-per-gene numbers because there are always two terminal (i.e., the amino and carboxyl) regions - independent of the length of sequences.

  17. Skipping Syntactically Illegal "the" Previews: The Role of Predictability

    Science.gov (United States)

    Abbott, Matthew J.; Angele, Bernhard; Ahn, Y. Danbi; Rayner, Keith

    2015-01-01

    Readers tend to skip words, particularly when they are short, frequent, or predictable. Angele and Rayner (2013) recently reported that readers are often unable to detect syntactic anomalies in parafoveal vision. In the present study, we manipulated target word predictability to assess whether contextual constraint modulates…

  18. Effect of skipping breakfast on subsequent energy intake.

    Science.gov (United States)

    Levitsky, David A; Pacanowski, Carly R

    2013-07-02

    The objective was to examine the effect of consuming breakfast on subsequent energy intake. Participants who habitually ate breakfast and those who skipped breakfast were recruited for two studies. Using a randomized crossover design, the first study examined the effect of having participants consume either (a) no breakfast, (b) a high carbohydrate breakfast (335 kcals), or (c) a high fiber breakfast (360 kcals) on three occasions and measured ad libitum intake at lunch. The second study again used a randomized crossover design but with a larger, normal carbohydrate breakfast consumed ad libtum. Intake averaged 624 kcals and subsequent food intake was measured throughout the day. Participants ate only foods served from the Cornell Human Metabolic Research Unit where all foods were weighed before and after consumption. In the first study, neither eating breakfast nor the kind of breakfast consumed had an effect on the amount consumed at lunch despite a reduction in hunger ratings. In the second study, intake at lunch as well as hunger ratings were significantly increased after skipping breakfast (by 144 kcal), leaving a net caloric deficit of 408 kcal by the end of the day. These data are consistent with published literature demonstrating that skipping a meal does not result in accurate energy compensation at subsequent meals and suggests that skipping breakfast may be an effective means to reduce daily energy intake in some adults.

  19. The relation between breakfast skipping and school performance in adolescents

    NARCIS (Netherlands)

    Boschloo, Annemarie; Ouwehand, Carolijn; Dekker, Sanne; Lee, Nikki; De Groot, Renate; Krabbendam, Lydia; Jolles, Jelle

    2012-01-01

    Boschloo, A., Ouwehand, C., Dekker, S., Lee, N., De Groot, R., Krabbendam, L., & Jolles, J. (2012). The relation between breakfast skipping and school performance in adolescents. Mind, Brain, and Education, 6(2), 81-88. doi:10.1111/j.1751-228x.2012.01138.x

  20. A missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient.

    Science.gov (United States)

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Bartolo, C; Sedra, M S; Western, L M; Mendell, J R

    1993-08-01

    About two thirds of Duchenne muscular dystrophy (DMD) patients have either gene deletions or duplications. The other DMD cases are most likely the result of point mutations that cannot be easily identified by current strategies. Utilizing a heteroduplex technique and direct sequencing of amplified products, we screened our nondeletion/duplication DMD population for point mutations. We now describe what we believe to be the first dystrophin missense mutation in a DMD patient. The mutation results in the substitution of an evolutionarily conserved leucine to arginine in the actin-binding domain. The patient makes a dystrophin protein which is properly localized and is present at a higher level than is observed in DMD patients. This suggests that an intact actin-binding domain is necessary for protein stability and essential for function.

  1. Mutations in the unc-52 gene responsible for body wall muscle defects in adult Caenorhabditis elegans are located in alternatively spliced exons

    Energy Technology Data Exchange (ETDEWEB)

    Rogalski, T.M.; Gilchrist, E.J.; Mullen, G.P. [Univ. of British, Columbia, Vancouver (Canada)] [and others

    1995-01-01

    The unc-52 gene in Caenorhabditis elegans produces several large proteins that function in the basement membrane underlying muscle cells. Mutations in this gene result in defects in myofilament assembly and in the attachment of the myofilament lattice to the muscle cell membrane. The st549 and ut111 alleles of unc-52 produce a lethal (Pat) terminal phenotype whereas the e444, e669, e998, e1012 and e1421 mutations result in viable, paralyzed animals. We have identified the sequence alterations responsible for these mutant phenotypes. The st549 allele has a premature stop codon in exon 7 that should result in the complete elimination of unc-52 gene function, and the ut111 allele has a Tc1 transposon inserted into the second exon of the gene. The five remaining mutations are clustered in a small interval containing three adjacent, alternatively spliced exons (16, 17 and 18). These mutations affect some, but not all of the unc-52-encoded proteins. Thirteen intragenic revertants of the e669, e998, e1012 and e1421 alleles have also been sequenced. The majority of these carry the original mutation plus a G to A transition in the conserved splice acceptor site of the affected exon. This result suggests that reversion of the mutant phenotype in these strains may be the result of exon-skipping. 38 refs., 6 figs., 2 tabs.

  2. A novel point mutation within the EDA gene causes an exon dropping in mature RNA in Holstein Friesian cattle breed affected by X-linked anhidrotic ectodermal dysplasia

    Directory of Open Access Journals (Sweden)

    Pariset Lorraine

    2011-07-01

    Full Text Available Abstract Background X-linked anhidrotic ectodermal dysplasia is a disorder characterized by abnormal development of tissues and organs of ectodermal origin caused by mutations in the EDA gene. The bovine EDA gene encodes the ectodysplasin A, a membrane protein expressed in keratinocytes, hair follicles and sweat glands, which is involved in the interactions between cell and cell and/or cell and matrix. Four mutations causing ectodermal dysplasia in cattle have been described so far. Results We identified a new single nucleotide polymorphism (SNP at the 9th base of exon 8 in the EDA gene in two calves of Holstein Friesian cattle breed affected by ectodermal dysplasia. This SNP is located in the exonic splicing enhancer (ESEs recognized by SRp40 protein. As a consequence, the spliceosome machinery is no longer able to recognize the sequence as exonic and causes exon skipping. The mutation determines the deletion of the entire exon (131 bp in the RNA processing, causing a severe alteration of the protein structure and thus the disease. Conclusion We identified a mutation, never described before, that changes the regulation of alternative splicing in the EDA gene and causes ectodermal dysplasia in cattle. The analysis of the SNP allows the identification of carriers that can transmit the disease to the offspring. This mutation can thus be exploited for a rational and efficient selection of unequivocally healthy cows for breeding.

  3. Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

    Science.gov (United States)

    Barnabei, Matthew S; Metzger, Joseph M

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2) mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

  4. Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

    Directory of Open Access Journals (Sweden)

    Matthew S Barnabei

    Full Text Available Duchenne muscular dystrophy (DMD is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from β-sarcoglycan-null and laminin-α(2 mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

  5. The Dystrophin-Glycoprotein Complex in the Prevention of Muscle Damage

    OpenAIRE

    2011-01-01

    Muscular dystrophies are genetically diverse but share common phenotypic features of muscle weakness, degeneration, and progressive decline in muscle function. Previous work has focused on understanding how disruptions in the dystrophin-glycoprotein complex result in muscular dystrophy, supporting a hypothesis that the muscle sarcolemma is fragile and susceptible to contraction-induced injury in multiple forms of dystrophy. Although benign in healthy muscle, contractions in dystrophic muscle ...

  6. Expression of dystrophin-glycoprotein complex at the skeletal muscle sarcolemma in Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Lei ZHAO

    2015-07-01

    Full Text Available Background  Eccentric exercise or high tension exercise could cause damage to skeletal muscle structure, resulting in deficiency of dystrophin and secondary loss of dystrophin-glycoprotein complex (DGC from the sarcolemma, which indicated that down-regulation of dystrophin was one of the key points of skeletal muscle injury from eccentric exercise. Duchenne muscular dystrophy (DMD is caused by mutations of DMD gene, resulting in the absence of dystrophin, which means that skeletal muscles of DMD patients after birth are in the natural state of actual path of force transmission which carried high tension from eccentric exercise. This paper investigated systematically whether expression of DGC is associated with progressive muscle weakness in natural history of DMD, and analyzed the expression of DGC at the sarcolemma of 197 confirmed DMD cases (9 days-12 years old.  Methods  The expression of α- and β-dystroglycan (DG, α-, β-, γ- and δ-sarcoglycan (SG and syntrophin at the sarcolemma of DMD patients was analyzed by immunofluorescent staining.  Results  The study showed that there was no relationship between lack of proteins and progressive muscle weakness with increasing age, although expression of α- and β-DG, α-, β-, γ- and δ-SG and syntrophin at the sarcolemma at different stages of 197 DMD patients (9 days-12 years old had different degrees of deficiency.  Conclusions  Deficiency of DGC may occur before birth and DMD patients were recommended to avoid further damage to skeletal muscles from eccentric exercise and high-resistance movement in activities of daily life and rehabilitation training. DOI: 10.3969/j.issn.1672-6731.2015.06.006

  7. Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins

    DEFF Research Database (Denmark)

    Moghadaszadeh, Behzad; Albrechtsen, Reidar; Guo, Ling T;

    2003-01-01

    , and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of alpha 7B integrin and utrophin, the functional homolog of dystrophin....... The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans....

  8. Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy

    NARCIS (Netherlands)

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A.; Williams, Donna L.; Deuss, Peter; Gait, Michael J.

    2015-01-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the

  9. A Novel Nonsense Mutation in CEP290 Induces Exon Skipping and Leads to a Relatively Mild Retinal Phenotype

    NARCIS (Netherlands)

    Littink, Karin W.; Pott, Jan-Willem R.; Collin, Rob W. J.; Kroes, Hester Y.; Verheij, Joke B. G. M.; Blokland, Ellen A. W.; Miro, Marta de Castro; Hoyng, Carel B.; Klaver, Caroline C. W.; Koenekoop, Robert K.; Rohrschneider, Klaus; Cremers, Frans P. M.; van den Born, L. Ingeborgh; den Hollander, Anneke I.

    2010-01-01

    PURPOSE. To identify the genetic defect in a family with variable retinal phenotypes. The proband had a diagnosis of Leber congenital amaurosis (LCA), whereas her two cousins had an early-onset severe retinal dystrophy (EOSRD) with useful vision. A distant family member had retinitis pigmentosa (RP)

  10. Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy

    NARCIS (Netherlands)

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A.; Williams, Donna L.; Deuss, Peter; Gait, Michael J.

    2015-01-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELecti

  11. In silico analyses of dystrophin Dp40 cellular distribution, nuclear export signals and structure modeling

    Directory of Open Access Journals (Sweden)

    Alejandro Martínez-Herrera

    2015-09-01

    Full Text Available Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids 93LEQEHNNLV101 and 168LLLHDSIQI176 could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled “EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40” (J. Aragón et al. Neurosci. Lett. 600 (2015 115–120 [1].

  12. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9.

    Science.gov (United States)

    Chen, Yongchang; Zheng, Yinghui; Kang, Yu; Yang, Weili; Niu, Yuyu; Guo, Xiangyu; Tu, Zhuchi; Si, Chenyang; Wang, Hong; Xing, Ruxiao; Pu, Xiuqiong; Yang, Shang-Hsun; Li, Shihua; Ji, Weizhi; Li, Xiao-Jiang

    2015-07-01

    CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy.

  13. Immobilization and therapeutic passive stretching generate thickening and increase the expression of laminin and dystrophin in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Cação-Benedini, L.O.; Ribeiro, P.G. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Medicina e Reabilitação do Aparelho Locomotor, Departamento de Biomecânica, Ribeirão Preto, SP, Brasil, Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Prado, C.M.; Chesca, D.L. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Patologia, Ribeirão Preto, SP, Brasil, Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Mattiello-Sverzut, A.C. [Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Medicina e Reabilitação do Aparelho Locomotor, Departamento de Biomecânica, Ribeirão Preto, SP, Brasil, Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2014-05-09

    Extracellular matrix and costamere proteins transmit the concentric, isometric, and eccentric forces produced by active muscle contraction. The expression of these proteins after application of passive tension stimuli to muscle remains unknown. This study investigated the expression of laminin and dystrophin in the soleus muscle of rats immobilized with the right ankle in plantar flexion for 10 days and subsequent remobilization, either by isolated free movement in a cage or associated with passive stretching for up to 10 days. The intensity of the macrophage response was also evaluated. One hundred and twenty-eight female Wistar rats were divided into 8 groups: free for 10 days; immobilized for 10 days; immobilized/free for 1, 3, or 10 days; or immobilized/stretched/free for 1, 3, or 10 days. After the experimental procedures, muscle tissue was processed for immunofluorescence (dystrophin/laminin/CD68) and Western blot analysis (dystrophin/laminin). Immobilization increased the expression of dystrophin and laminin but did not alter the number of macrophages in the muscle. In the stretched muscle groups, there was an increase in dystrophin and the number of macrophages after 3 days compared with the other groups; dystrophin showed a discontinuous labeling pattern, and laminin was found in the intracellular space. The amount of laminin was increased in the muscles treated by immobilization followed by free movement for 10 days. In the initial stages of postimmobilization (1 and 3 days), an exacerbated macrophage response and an increase of dystrophin suggested that the therapeutic stretching technique induced additional stress in the muscle fibers and costameres.

  14. Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy.

    Directory of Open Access Journals (Sweden)

    Johann Böhm

    2013-06-01

    Full Text Available Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM, a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM. However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD. Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies.

  15. Alternative splicing of exon 17 and a missense mutation in exon 20 of the insulin receptor gene in two brothers with a novel syndrome of insulin resistance (congenital fiber-type disproportion myopathy).

    Science.gov (United States)

    Vorwerk, P; Christoffersen, C T; Müller, J; Vestergaard, H; Pedersen, O; De Meyts, P

    1999-01-01

    The insulin receptor (IR) in two brothers with a rare syndrome of congenital muscle fiber type disproportion myopathy (CFTDM) associated with diabetes and severe insulin resistance was studied. By direct sequencing of Epstein-Barr virus-transformed lymphocytes both patients were found to be compound heterozygotes for mutations in the IR gene. The maternal allele was alternatively spliced in exon 17 due to a point mutation in the -1 donor splice site of the exon. The abnormal skipping of exon 17 shifts the amino acid reading frame and leads to a truncated IR, missing the entire tyrosine kinase domain. In the correct spliced variant, the point mutation is silent and results in a normally translated IR. The paternal allele carries a missense mutation in the tyrosine kinase domain. All three cDNA variants were present in the lymphocytes of the patients. Purified IR from 293 cells overexpressing either of the two mutated receptors lacked basal or stimulated IR beta-subunit autophosphorylation. A third brother who inherited both normal alleles has an normal muscle phenotype and insulin sensitivity, suggesting a direct linkage of these IR mutations with the CFTDM phenotype.

  16. Two novel exonic point mutations in HEXA identified in a juvenile Tay-Sachs patient: role of alternative splicing and nonsense-mediated mRNA decay.

    Science.gov (United States)

    Levit, A; Nutman, D; Osher, E; Kamhi, E; Navon, R

    2010-06-01

    We have identified three mutations in the beta-hexoseaminidase A (HEXA) gene in a juvenile Tay-Sachs disease (TSD) patient, which exhibited a reduced level of HEXA mRNA. Two mutations are novel, c.814G>A (p.Gly272Arg) and c.1305C>T (p.=), located in exon 8 and in exon 11, respectively. The third mutation, c.1195A>G (p.Asn399Asp) in exon 11, has been previously characterized as a common polymorphism in African-Americans. Hex A activity measured in TSD Glial cells, transfected with HEXA cDNA constructs bearing these mutations, was unaltered from the activity level measured in normal HEXA cDNA. Analysis of RT-PCR products revealed three aberrant transcripts in the patient, one where exon 8 was absent, one where exon 11 was absent and a third lacking both exons 10 and 11. All three novel transcripts contain frameshifts resulting in premature termination codons (PTCs). Transfection of mini-gene constructs carrying the c.814G>A and c.1305C>T mutations proved that the two mutations result in exon skipping. mRNAs that harbor a PTC are detected and degraded by the nonsense-mediated mRNA decay (NMD) pathway to prevent synthesis of abnormal proteins. However, although NMD is functional in the patient's fibroblasts, aberrant transcripts are still present. We suggest that the level of correctly spliced transcripts as well as the efficiency in which NMD degrade the PTC-containing transcripts, apparently plays an important role in the phenotype severity of the unique patient and thus should be considered as a potential target for drug therapy.

  17. In vivo proof-of-concept of removal of the huntingtin caspase cleavage motif-encoding exon 12 approach in the YAC128 mouse model of Huntington's disease.

    Science.gov (United States)

    Casaca-Carreira, João; Toonen, Lodewijk J A; Evers, Melvin M; Jahanshahi, Ali; van-Roon-Mom, Willeke M C; Temel, Yasin

    2016-12-01

    Huntington's disease (HD) is a progressive autosomal dominant disease, caused by a CAG repeat expansion in the HTT gene, resulting in an expanded polyglutamine stretch at the N-terminal of the huntingtin protein. An important event in HD pathogenesis appears to be the proteolysis of the mutant protein, which forms N-terminal huntingtin fragments. These fragments form insoluble aggregates and are found in nuclei and cytoplasm of affected neurons where they interfere with normal cell functioning. Important cleavage sites are encoded by exon 12 of HTT. A novel approach is Htt protein modification through exon skipping, which has recently been proven effective both in vitro and in vivo. Here we report proof-of-concept of AON 12.1 in vivo using the YAC128 mouse model of HD. Our results support and encourage future longitudinal studies exploring the therapeutic effects of sustained infusions in the YAC128 mouse model.

  18. Exon-centric regulation of pyruvate kinase M alternative splicing via mutually exclusive exons

    Institute of Scientific and Technical Information of China (English)

    Zhenxun Wang; Deblina Chatterjee; Hyun Yong Jeon; Martin Akerman; Matthew G. Vander Heiden; Lewis C. Cantley; Adrian R. Krainer

    2012-01-01

    Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth.However,the cancer-specific alternative splicing regulation of PK-M is not completely understood.Here,we demonstrate that PK-M is regulated by reciprocal affects on the mutually exclusive exons 9 and 10,such that exon 9 is repressed and exon 10 is activated in cancer cells.Strikingly,exonic,rather than intronic,cis-elements are key determinants ef PK-M splicing isoform ratios.Using a systematic sub-exonic duplication approach,we identify a potent exonlc splicing enhancer in exon 10,which differs from its homologous counterpart in exon 9 by only two nucleotides.We identify SRSF3 as one of the cognate factors,and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism.These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect,and also have implications for other genes with a similar pattern of alternative splicing.

  19. Variable Block Carry Skip Logic using Reversible Gates

    CERN Document Server

    Islam, Md Rafiqul; Karim, Muhammad Rezaul; Mahmud, Abdullah Al; Babu, Hafiz Md Hasan

    2010-01-01

    Reversible circuits have applications in digital signal processing, computer graphics, quantum computation and cryptography. In this paper, a generalized k*k reversible gate family is proposed and a 3*3 gate of the family is discussed. Inverter, AND, OR, NAND, NOR, and EXOR gates can be realized by this gate. Implementation of a full-adder circuit using two such 3*3 gates is given. This full-adder circuit contains only two reversible gates and produces no extra garbage outputs. The proposed full-adder circuit is efficient in terms of gate count, garbage outputs and quantum cost. A 4-bit carry skip adder is designed using this full-adder circuit and a variable block carry skip adder is discussed. Necessary equations required to evaluate these adder are presented.

  20. Skipping breakfast adversely affects menstrual disorders in young college students.

    Science.gov (United States)

    Fujiwara, Tomoko; Sato, Natsuyo; Awaji, Hiroyo; Sakamoto, Hiroko; Nakata, Rieko

    2009-01-01

    In the present study we conducted a questionnaire survey to examine the relationship between dietary habits and menstrual disorders in young women. Subjects were recruited from 315 college students and were classified as: Group I, eating breakfast; Group II, skipping breakfast; Group III, not eating fast foods; Group IV, eating fast foods; Group V, not eating processed foods; and Group VI, eating processed foods. The intensity of dysmenorrhea was scored using three grades. All participants were further divided into groups based on having regular or irregular menstruation, having premenstrual symptoms or not, and self-perception of good or poor general health. General health was poor in Groups II and VI, and dysmenorrhea scores were high in Groups II, IV and VI. The incidence of irregular menses was also high in Group II. However, there was no apparent relation between premenstrual symptoms and dietary habits. These findings suggest that skipping breakfast adversely affects menstrual disorders in young college students.

  1. Potential applications of skip SMV with thrust engine

    Science.gov (United States)

    Wang, Weilin; Savvaris, Al

    2016-11-01

    This paper investigates the potential applications of Space Maneuver Vehicles (SMV) with skip trajectory. Due to soaring space operations over the past decades, the risk of space debris has considerably increased such as collision risks with space asset, human property on ground and even aviation. Many active debris removal methods have been investigated and in this paper, a debris remediation method is first proposed based on skip SMV. The key point is to perform controlled re-entry. These vehicles are expected to achieve a trans-atmospheric maneuver with thrust engine. If debris is released at altitude below 80 km, debris could be captured by the atmosphere drag force and re-entry interface prediction accuracy is improved. Moreover if the debris is released in a cargo at a much lower altitude, this technique protects high value space asset from break up by the atmosphere and improves landing accuracy. To demonstrate the feasibility of this concept, the present paper presents the simulation results for two specific mission profiles: (1) descent to predetermined altitude; (2) descent to predetermined point (altitude, longitude and latitude). The evolutionary collocation method is adopted for skip trajectory optimization due to its global optimality and high-accuracy. This method is actually a two-step optimization approach based on the heuristic algorithm and the collocation method. The optimal-control problem is transformed into a nonlinear programming problem (NLP) which can be efficiently and accurately solved by the sequential quadratic programming (SQP) procedure. However, such a method is sensitive to initial values. To reduce the sensitivity problem, genetic algorithm (GA) is adopted to refine the grids and provide near optimum initial values. By comparing the simulation data from different scenarios, it is found that skip SMV is feasible in active debris removal and the evolutionary collocation method gives a truthful re-entry trajectory that satisfies the

  2. Delay Efficient 32-Bit Carry-Skip Adder

    OpenAIRE

    Yu Shen Lin; Damu Radhakrishnan

    2008-01-01

    The design of a 32-bit carry-skip adder to achieve minimum delay is presented in this paper. A fast carry look-ahead logic using group generate and group propagate functions is used to speed up the performance of multiple stages of ripple carry adders. The group generate and group propagate functions are generated in parallel with the carry generation for each block. The optimum block sizes are decided by considering the critical path into account. The new architecture ...

  3. An Index Based Skip Search Multiple Pattern Matching Algorithm

    OpenAIRE

    Raju Bhukya; Balram Parmer,; Anand Kulkarni

    2011-01-01

    DNA Pattern matching, the problem of finding sub sequences within a long DNA sequence has many applications in computational biology. As the sequences can be long, matching can be an expensive operation, especially as approximate matching is allowed. Searching DNA related data is a common activity for molecular biologists. In this paper we explore the applicability of a new pattern matching technique called Index based Skip Search Multiple Pattern matching algorithm (ISMPM), for DNA sequences...

  4. An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

    Directory of Open Access Journals (Sweden)

    Dario Balestra

    2016-01-01

    Full Text Available In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1 can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5′ (c.519A > G or 3′ (c.392-8T > G splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5′ss and hFIX-8G3′ss splicing-competent human factor IX (hFIX cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5′ss, 1.0 ± 0.5 µg/ml; hFIX-8G3′ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml, leading to a striking shortening (from ≃100 seconds of untreated mice to ≃80 seconds of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

  5. Kinematic characteristics of motor patterns in rope skipping

    Directory of Open Access Journals (Sweden)

    Luiz Henrique da Silva

    2009-09-01

    Full Text Available Rope skipping seems to be an easy task to be performed. However, careful analysis of this motor skill shows how complex the execution of this task is. The objective of this study was to examine kinematic variables of jump patterns as a function of skipping frequency. Eight male university students performed a sequence of 30 rope jumps using two jump patterns (alternating support of the feet and simultaneous support of the feet at three skipping frequencies (1.5, 1.7,1.9 Hz. Frequencies were determined with a digital metronome and the rope was turned by the student himself. Rope jumping performance was recorded with two digital cameras for 3Danalysis. Passive markers were attached to the rope and to the ankle, knee and hip joints forcollection of the following dependent variables: continuous relative phase, time interval betweenthe loss of contact of the feet with the ground and cross of the rope under the feet of the volunteer,jump height, and rope height. ANOVA showed that for the pattern with alternating support ofthe feet the jump is executed at a lower height. In addition, analysis of the time interval revealeda delay in the withdrawal of the feet for crossing the rope in the case of the jump pattern with simultaneous support of the feet.

  6. Leptin Level and Skipping Breakfast: The National Health and Nutrition Examination Survey III (NHANES III)

    OpenAIRE

    Keiko Asao; Amandine Sambira Marekani; Jessica VanCleave; Amy E. Rothberg

    2016-01-01

    Skipping breakfast is a common dietary habit considered to be unhealthy. However, the mechanisms underlying skipping breakfast have not been fully explored. Leptin is a hormone that regulates food intake and energy storage and secretes in a diurnal rhythm with lowest levels in the morning. We examined the association between the serum leptin level and skipping breakfast in 5714 adults in the U.S. National Health and Nutrition Examination Survey III, 1988–1994. We defined breakfast as any food...

  7. Faster exon assembly by sparse spliced alignment

    CERN Document Server

    Tiskin, Alexander

    2007-01-01

    Assembling a gene from candidate exons is an important problem in computational biology. Among the most successful approaches to this problem is \\emph{spliced alignment}, proposed by Gelfand et al., which scores different candidate exon chains within a DNA sequence of length $m$ by comparing them to a known related gene sequence of length n, $m = \\Theta(n)$. Gelfand et al.\\ gave an algorithm for spliced alignment running in time O(n^3). Kent et al.\\ considered sparse spliced alignment, where the number of candidate exons is O(n), and proposed an algorithm for this problem running in time O(n^{2.5}). We improve on this result, by proposing an algorithm for sparse spliced alignment running in time O(n^{2.25}). Our approach is based on a new framework of \\emph{quasi-local string comparison}.

  8. Social Inequalities in Young Children's Meal Skipping Behaviors: The Generation R Study.

    Directory of Open Access Journals (Sweden)

    Anne I Wijtzes

    Full Text Available Regular meal consumption is considered an important aspect of a healthy diet. While ample evidence shows social inequalities in breakfast skipping among adolescents, little is known about social inequalities in breakfast skipping and skipping of other meals among young school-aged children. Such information is crucial in targeting interventions aimed to promote a healthy diet in children.We examined data from 4704 ethnically diverse children participating in the Generation R Study, a population-based prospective cohort study in Rotterdam, the Netherlands. Information on family socioeconomic position (SEP, ethnic background, and meal skipping behaviors was assessed by parent-reported questionnaire when the child was 6 years old. Multiple logistic regression analyses were performed to assess the associations of family SEP (educational level, household income, employment status, family composition and ethnic background with meal skipping behaviors, using high SEP children and native Dutch children as reference groups.Meal skipping prevalence ranged from 3% (dinner to 11% (lunch. The prevalence of meal skipping was higher among low SEP children and ethnic minority children. Maternal educational level was independently associated with breakfast skipping ([low maternal educational level] OR: 2.21; 95% CI: 1.24,3.94. Paternal educational level was independently associated with lunch skipping ([low paternal educational level] OR: 1.53; 95% CI: 1.06,2.20 and dinner skipping ([mid-high paternal educational level] OR: 0.39; 95% CI: 0.20,0.76. Household income was independently associated with breakfast skipping ([low income] OR: 2.43, 95% CI: 1.40,4.22 and dinner skipping ([low income] OR: 2.44; 95% CI: 1.22,4.91. In general, ethnic minority children were more likely to skip breakfast, lunch, and dinner compared with native Dutch children. Adjustment for family SEP attenuated the associations of ethnic minority background with meal skipping behaviors

  9. Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes.

    Science.gov (United States)

    Sabourin, Jessica; Harisseh, Rania; Harnois, Thomas; Magaud, Christophe; Bourmeyster, Nicolas; Déliot, Nadine; Constantin, Bruno

    2012-12-01

    In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.

  10. A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Chantal Beekman

    Full Text Available Duchenne muscular dystrophy (DMD is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2-17% and intra-assay precision, CV 2-10%. Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.

  11. Breakfast skipping and body mass index among adolescents in Greece: whether an association exists depends on how breakfast skipping is defined.

    Science.gov (United States)

    Dialektakou, Kiranni D; Vranas, Peter B M

    2008-09-01

    Many studies have found an association between breakfast skipping and either body mass index (BMI; calculated as kg/m(2)) or overweight/obesity among adolescents, but several studies have found no association. This cross-sectional study investigated the hypothesis that this discrepancy is partly due to three differences in methodology. First, some studies have examined BMI, but other studies have examined overweight/obesity. Second, some studies have controlled for potential confounders, but other studies have not. Third, different studies have used different definitions of breakfast skipping. This study examined both the relationship between breakfast skipping and BMI and the relationship between breakfast skipping and overweight/obesity, compared unadjusted results with results adjusted for potential confounders, and compared results for 24 definitions of breakfast skipping. The sample consisted of 811 students at high schools in Piraeus, Greece, who completed a questionnaire and had their height and weight measured. The results supported this hypothesis. First, fewer breakfast-skipping variables were associated with BMI than with overweight/obesity. Second, fewer associations were found when controlling than when not controlling for potential confounders. Third, fewer associations were found for variables corresponding to some definitions of breakfast skipping than for variables corresponding to other definitions.

  12. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

    Directory of Open Access Journals (Sweden)

    Erik van der Wal

    2017-06-01

    Full Text Available The most common variant causing Pompe disease is c.-32-13T>G (IVS1 in the acid α-glucosidase (GAA gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity.

  13. Effect of exonic splicing regulation on synonymous codon usage in alternatively spliced exons of Dscam

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    Takahashi Aya

    2009-08-01

    Full Text Available Abstract Background Synonymous codon usage is typically biased towards translationally superior codons in many organisms. In Drosophila, genomic data indicates that translationally optimal codons and splice optimal codons are mostly mutually exclusive, and adaptation to translational efficiency is reduced in the intron-exon boundary regions where potential exonic splicing enhancers (ESEs reside. In contrast to genomic scale analyses on large datasets, a refined study on a well-controlled set of samples can be effective in demonstrating the effects of particular splice-related factors. Down syndrome cell adhesion molecule (Dscam has the largest number of alternatively spliced exons (ASEs known to date, and the splicing frequency of each ASE is accessible from the relative abundance of the transcript. Thus, these ASEs comprise a unique model system for studying the effect of splicing regulation on synonymous codon usage. Results Codon Bias Indices (CBI in the 3' boundary regions were reduced compared to the rest of the exonic regions among 48 and 33 ASEs of exon 6 and 9 clusters, respectively. These regional differences in CBI were affected by splicing frequency and distance from adjacent exons. Synonymous divergence levels between the 3' boundary region and the remaining exonic region of exon 6 ASEs were similar. Additionally, another sensitive comparison of paralogous exonic regions in recently retrotransposed processed genes and their parental genes revealed that, in the former, the differences in CBI between what were formerly the central regions and the boundary regions gradually became smaller over time. Conclusion Analyses of the multiple ASEs of Dscam allowed direct tests of the effect of splice-related factors on synonymous codon usage and provided clear evidence that synonymous codon usage bias is restricted by exonic splicing signals near the intron-exon boundary. A similar synonymous divergence level between the different exonic

  14. Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery

    Directory of Open Access Journals (Sweden)

    Xuan Guan

    2014-03-01

    Full Text Available The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD. Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs. USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

  15. Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise, Neuromuscular Junctions, and Brains

    Directory of Open Access Journals (Sweden)

    Bailey Nichols

    2015-07-01

    Full Text Available Dystrophin-glycoprotein complex (DGC is an important structural unit in skeletal muscle that connects the cytoskeleton (f-actin of a muscle fiber to the extracellular matrix (ECM. Several muscular dystrophies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophies (dystroglycanopathies, and limb-girdle muscular dystrophies (sarcoglycanopathies, are caused by mutations in the different DGC components. Although many early studies indicated DGC plays a crucial mechanical role in maintaining the structural integrity of skeletal muscle, recent studies identified novel roles of DGC. Beyond a mechanical role, these DGC members play important signaling roles and act as a scaffold for various signaling pathways. For example, neuronal nitric oxide synthase (nNOS, which is localized at the muscle membrane by DGC members (dystrophin and syntrophins, plays an important role in the regulation of the blood flow during exercise. DGC also plays important roles at the neuromuscular junction (NMJ and in the brain. In this review, we will focus on recently identified roles of DGC particularly in exercise and the brain.

  16. Current understanding of dystrophin-related muscular dystrophy and therapeutic challenges ahead

    Institute of Scientific and Technical Information of China (English)

    ZHOU Guang-qian; XIE Hui-qi; ZHANG Su-zhen; YANG Zhi-ming

    2006-01-01

    Objective To review the recent research progress in dystrophin-related muscular dystrophy includes X-linked hereditary Duchenne and Becker muscular dystrophies (DMD and BMD).Data sources Information included in this article was identified by searches of PUBMED and other online resources using the key terms DMD, dystrophin, mutations, animal models, pathophysiology, gene expression, stem cells, gene therapy, cell therapy, and pharmacological.Study selection Mainly original milestone articles and timely reviews written by major pioneer investigators of the field were selected.Results The key issues related to the genetic basis and pathophysiological factors of the diseases were critically addressed. The availabilities and advantages of various animal models for the diseases were described. Major molecular and cellular therapeutic approaches were also discussed, many of which have indeed exhibited some success in pre-clinical studies but at the same time encountered a number of technical hurdles, including the efficient and systemic delivery of a functional gene and myogenic precursor/stem cells to repair genetic defects.Conclusions Further understanding of pathophysiological mechanisms at molecular levels and regenerative properites of myogenic precursor/stem cells will promote the development of multiple therapeutic strategies. The combined use of multiple strategies may represent the major challenge as well as the greatest hope for the therapy of these diseases in coming years.

  17. Fetal skeletal muscle progenitors have regenerative capacity after intramuscular engraftment in dystrophin deficient mice.

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    Hiroshi Sakai

    Full Text Available Muscle satellite cells (SCs are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs isolated from Pax3(GFP/+ embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3(GFP/+ mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease.

  18. Membrane Sealant Poloxamer P188 Protects Against Isoproterenol Induced Cardiomyopathy in Dystrophin Deficient Mice

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    Sali Arpana

    2011-05-01

    Full Text Available Abstract Background Cardiomyopathy in Duchenne muscular dystrophy (DMD is an increasing cause of death in patients. The absence of dystrophin leads to loss of membrane integrity, cell death and fibrosis in cardiac muscle. Treatment of cardiomyocyte membrane instability could help prevent cardiomyopathy. Methods Three month old female mdx mice were exposed to the β1 receptor agonist isoproterenol subcutaneously and treated with the non-ionic tri-block copolymer Poloxamer P188 (P188 (460 mg/kg/dose i.p. daily. Cardiac function was assessed using high frequency echocardiography. Tissue was evaluated with Evans Blue Dye (EBD and picrosirius red staining. Results BL10 control mice tolerated 30 mg/kg/day of isoproterenol for 4 weeks while death occurred in mdx mice at 30, 15, 10, 5 and 1 mg/kg/day within 24 hours. Mdx mice tolerated a low dose of 0.5 mg/kg/day. Isoproterenol exposed mdx mice showed significantly increased heart rates (p Conclusions This model suggests that chronic intermittent intraperitoneal P188 treatment can prevent isoproterenol induced cardiomyopathy in dystrophin deficient mdx mice.

  19. Breakfast skipping is associated with cyberbullying and school bullying victimization:a school-based cross-sectional study

    OpenAIRE

    Sampasa-Kanyinga, Hugues; Roumeliotis, Paul; Farrow, Claire V.; Shi, Yuanfeng F.

    2014-01-01

    Breakfast skipping is a health concern that has well-known negative consequences physically and psychologically. It is therefore important to understand why children skip breakfast. The purpose of this study was to establish whether the experience of bullying and cyberbullying impacts upon breakfast skipping and to further evaluate whether the inability for youths to cope with bullying victimization affects their mental health (depression), and in turn predicts breakfast skipping. Data were o...

  20. Word skipping: effects of word length, predictability, spelling and reading skill.

    Science.gov (United States)

    Slattery, Timothy J; Yates, Mark

    2017-08-31

    Readers eyes often skip over words as they read. Skipping rates are largely determined by word length; short words are skipped more than long words. However, the predictability of a word in context also impacts skipping rates. Rayner, Slattery, Drieghe and Liversedge (2011) reported an effect of predictability on word skipping for even long words (10-13 characters) that extend beyond the word identification span. Recent research suggests that better readers and spellers have an enhanced perceptual span (Veldre & Andrews, 2014). We explored whether reading and spelling skill interact with word length and predictability to impact word skipping rates in a large sample (N=92) of average and poor adult readers. Participants read the items from Rayner et al. (2011) while their eye movements were recorded. Spelling skill (zSpell) was assessed using the dictation and recognition tasks developed by Sally Andrews and colleagues. Reading skill (zRead) was assessed from reading speed (words per minute) and accuracy of three 120 word passages each with 10 comprehension questions. We fit linear mixed models to the target gaze duration data and generalized linear mixed models to the target word skipping data. Target word gaze durations were significantly predicted by zRead while, the skipping likelihoods were significantly predicted by zSpell. Additionally, for gaze durations, zRead significantly interacted with word predictability as better readers relied less on context to support word processing. These effects are discussed in relation to the lexical quality hypothesis and eye movement models of reading.

  1. Skipping breakfast is associated with reproductive dysfunction in post-adolescent female college students.

    Science.gov (United States)

    Fujiwara, Tomoko; Nakata, Rieko

    2010-12-01

    Although increasing attention has been paid to the adverse effects of skipping breakfast on quality of life, there are very few reports concerning the relationship between skipping breakfast and reproductive function. Therefore, we examined this issue by conducting a questionnaire survey of female college students aged from 18 to 20 years old. The 5 annual surveys of questionnaire demonstrated that the severity of dysmenorrhea was significantly higher in the population that skipped breakfast. The incidence of irregular menses was also higher in the population that skipped breakfast, although there was no difference in the incidence of premenstrual symptoms. The group that skipped breakfast showed a tendency to suffer from constipation. In addition, despite no difference in body mass index, there was a significantly higher incidence of a self-perception of poor general health among the group that skipped breakfast. These findings suggest that skipping breakfast is associated with menstrual disorders, and affects the physical condition of female college students who are undergoing post-adolescent maturation. Since these menstrual disorders may influence the quality of life of young women not only in the present but also in the future, skipping breakfast should be re-evaluated from the perspective of future reproductive function.

  2. Endoscopic Skipping of the Terminal Ileum in Pediatric Crohn Disease.

    Science.gov (United States)

    Mansuri, Ishrat; Fletcher, Joel G; Bruining, David H; Kolbe, Amy B; Fidler, Jeff L; Samuel, Sunil; Tung, Jeanne

    2017-06-01

    Pediatric small-bowel (SB) Crohn disease (CD) may be missed if the terminal ileum (TI) appears normal at endoscopy and SB imaging is not performed. We sought to estimate the prevalence and clinical characteristics of pediatric patients with CD and endoscopic skipping of the TI-that is, pediatric patients with active SB or upper gut inflammation and an endoscopically normal TI. This retrospective study included pediatric patients with CD who underwent both CT enterography (CTE) or MR enterography (MRE) and ileocolonoscopy within a 30-day period between July 2004 and April 2014. The physician global assessment was used as the reference standard for SB CD activity. Radiologists reviewed the CTE and MRE studies for inflammatory parameters; severity, length, and multifocality of SB inflammation; and the presence of penetrating complications. Of 170 patients who underwent ileal intubation, the TI was macroscopically normal or showed nonspecific inflammation in 73 patients (43%). Nearly half (36/73, 49%) of the patients with normal or nonspecific findings at ileocolonoscopy had radiologically active disease with a median length of SB involvement of 20 cm (range, 1 to > 100 cm). Seventeen (47%) of these patients had multifocal SB involvement and five (14%) had penetrating complications. Overall, endoscopic TI skipping was present in 43 (59%) patients with normal or nonspecific ileocolonoscopic findings: 20 with histologic inflammation (17 with positive imaging findings), 14 with inflammation at imaging only, and nine with proximal disease (upper gut, jejunum, or proximal ileum). There were no significant differences in the clinical parameters of the patients with and those without endoscopic TI skipping. Ileocolonoscopy may miss SB CD in pediatric patients that is due to isolated histologic, intramural, or proximal inflammation. Enterography is complementary to ileocolonoscopy in the evaluation of pediatric CD.

  3. Exon - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ontents Exons in variants Data file File name: astra_exon.zip File URL: ftp://ftp.biosciencedbc.jp/archive/a... About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Exon - ASTRA | LSDB Archive ...

  4. A case of an idiopathic massive osteolysis with skip lesions

    Energy Technology Data Exchange (ETDEWEB)

    Ozbayrak, Mustafa [Dept. of Radiology, Arnavutkoy State Hospital, Instanbul (Turkmenistan); Yilmaz, Mehmet Halit; Kantarci, Fatih; Ozer, Harun; Harmanci, Kemal; Babacan, Muharrem; Dervisoglu, Sergulen [Cerrahpasa Medical Faculty, Istanbul University, Istanbul (Turkmenistan)

    2013-12-15

    A patient with a 2-year history of pain in the left arm, and decreased strengths unrelieved by non-steroidal anti-inflammatory therapy, was being referred for repeating radiography. The radiologic examinations have demonstrated a unique pattern of non-contiguous osteolysis in the left elbow, proximal and distal radius, ulna, wrist, carpal bones, proximal and distal metacarpals and phalanges. Multi-site biopsies were being performed and confirmed the diagnosis of massive osteolysis. To our knowledge, this is the first case in which multifocal, non-contiguous osteolysis with skip lesions without associated nephropathy and without a hereditary pattern is being described in one extremity.

  5. mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration

    Directory of Open Access Journals (Sweden)

    Thomas C. Roberts

    2016-03-01

    Full Text Available Duchenne muscular dystrophy (DMD is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1 the determination of gene expression changes associated with dystrophic pathology, (2 identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3 investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis, and (4 prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO with the accession number GSE64420.

  6. Convergent and stereospecific synthesis of complex skipped polyenes and polyunsaturated fatty acids.

    Science.gov (United States)

    Macklin, Todd K; Micalizio, Glenn C

    2010-08-01

    Skipped polyenes (that is, 1,4-dienes and higher homologues) are stereodefined components of a vast array of biologically important natural products, including polyunsaturated fatty acids. Although widespread in nature, these architectures are generally considered to represent significant barriers to efficient chemical synthesis. Partial reduction of skipped poly-ynes provides a pathway to a subset of such structures, but general chemical methods for the preparation of skipped polyenes that contain varied stereochemistries and substitution patterns are lacking. Here, we describe a metal-promoted reductive cross-coupling reaction between vinylcyclopropanes and alkynes (or vinylsilanes) that provides stereoselective access to a diverse array of skipped polyenes through a process that establishes one C-C bond, generates up to three stereodefined alkenes, and can be used to introduce stereogenic centres at the central positions of the skipped polyene motif. We also demonstrate the significance of the present bond construction by preparing substituted and stereodefined polyunsaturated synthetic fatty acids.

  7. Eye movements and word skipping during reading: Effects of word length and predictability

    Science.gov (United States)

    Rayner, Keith; Slattery, Timothy J.; Drieghe, Denis; Liversedge, Simon P.

    2012-01-01

    The extent to which target words were predictable from prior context was varied: half of the target words were predictable and the other half were unpredictable. In addition, the length of the target word varied: the target words were short (4–6 letters), medium (7–9 letters), or long (10–12 letters). Length and predictability both yielded strong effects on the probability of skipping the target words and on the amount of time readers fixated the target words (when they were not skipped). However, there was no interaction in any of the measures examined for either skipping or fixation time. The results demonstrate that word predictability (due to contextual constraint) and word length have strong and independent influences on word skipping and fixation durations. Furthermore, since the long words extended beyond the word identification span, the data indicate that skipping can occur on the basis of partial information in relation to word identity. PMID:21463086

  8. Breakfast Skipping and Its Associated Factors among Undergraduates in a Public University in Kuala Lumpur.

    Science.gov (United States)

    Moy, F M; Johari, S; Ismail, Y; Mahad, R; Tie, F H; Wan Ismail, Wm A

    2009-09-01

    An analytical cross-sectional study was conducted in a public university in Kuala Lumpur among a random sample of 2665 undergraduates. The objective was to study the prevalence of breakfast skipping and its associated factors. Data collection was conducted via a self-administered pre-tested questionnaire. There were 43.5% male respondents, with Malays being the majority (58.3%). The prevalence of breakfast skipping was 29.2 (95% CI: 27.3 - 30.3)%. The factors significantly associated with breakfast skipping (pskipping dinner. As the respondents' age increased, their risk of breakfast skipping was lower (OR: 0.95; 0.89 - 0.99). Malays (OR: 1.94; 1.48 - 2.54), Indians (OR: 1.70; 1.08 - 2.66), and students from the Sabah and Sarawak indigenous communities (OR: 2.13; 1.37 - 3.33) were more likely to skip breakfast compared to their Chinese counterparts. Respondents who stayed in their own houses were also less likely to skip breakfast compared to those staying in hostel with meals catered (OR: 2.32; 1.39 - 3.84), hostel with cafeteria (OR: 2.92; 1.74 - 4.91) or in rented houses (OR: 2.08; 1.25 - 3.46). Respondents majoring in Arts and Economics had 1.40 (1.07 - 1.82) times risk of breakfast skipping compared to those majoring in Life Sciences. Those who skipped dinner too had twice the odds (1.47 - 2.77) of breakfast skipping. In conclusion the prevalence of breakfast skipping among the undergraduates of this university was moderately high. Health awareness campaigns or introduction of healthy eating guidelines should be initiated for the undergraduates as well as food caterers in campus. The policy and pricing of catered food in campus should also be reviewed.

  9. Bortezomib (PS-341 treatment decreases inflammation and partially rescues the expression of the dystrophin-glycoprotein complex in GRMD dogs.

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    Karla P C Araujo

    Full Text Available Golden retriever muscular dystrophy (GRMD is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs with the proteasome inhibitor bortezomib, and three were control dogs (CD. Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some

  10. Matrix metalloproteinase-2 ablation in dystrophin-deficient mdx muscles reduces angiogenesis resulting in impaired growth of regenerated muscle fibers.

    Science.gov (United States)

    Miyazaki, Daigo; Nakamura, Akinori; Fukushima, Kazuhiro; Yoshida, Kunihiro; Takeda, Shin'ichi; Ikeda, Shu-ichi

    2011-05-01

    Matrix metalloproteases (MMPs) are a family of endopeptidases classified into subgroups based on substrate preference in normal physiological processes such as embryonic development and tissue remodeling, as well as in various disease processes via degradation of extracellular matrix components. Among the MMPs, MMP-9 and MMP-2 have been reported to be up-regulated in skeletal muscles in the lethal X-linked muscle disorder Duchenne muscular dystrophy (DMD), which is caused by loss of dystrophin. A recent study showed that deletion of the MMP9 gene in mdx, a mouse model for DMD, improved skeletal muscle pathology and function; however, the role of MMP-2 in the dystrophin-deficient muscle is not well known. In this study, we aimed at verifying the role of MMP-2 in the dystrophin-deficient muscle by using mdx mice with genetic ablation of MMP-2 (mdx/MMP-2(-/-)). We found impairment of regenerated muscle fiber growth with reduction of angiogenesis in mdx/MMP-2(-/-) mice at 3 months of age. Expression of vascular endothelial growth factor-A (VEGF-A), an important angiogenesis-related factor, decreased in mdx/MMP-2(-/-) mice at 3 months of age. MMP-2 had not a critical role in the degradation of dystrophin-glycoprotein complex (DGC) components such as β-dystroglycan and β-sarcoglycan in the regeneration process of the dystrophic muscle. Accordingly, MMP-2 may be essential for growth of regenerated muscle fibers through VEGF-associated angiogenesis in the dystrophin-deficient skeletal muscle.

  11. Skipping Posterior Dynamic Transpedicular Stabilization for Distant Segment Degenerative Disease

    Directory of Open Access Journals (Sweden)

    Bilgehan Solmaz

    2012-01-01

    Full Text Available Objective. To date, there is still no consensus on the treatment of spinal degenerative disease. Current surgical techniques to manage painful spinal disorders are imperfect. In this paper, we aimed to evaluate the prospective results of posterior transpedicular dynamic stabilization, a novel surgical approach that skips the segments that do not produce pain. This technique has been proven biomechanically and radiologically in spinal degenerative diseases. Methods. A prospective study of 18 patients averaging 54.94 years of age with distant spinal segment degenerative disease. Indications consisted of degenerative disc disease (57%, herniated nucleus pulposus (50%, spinal stenosis (14.28%, degenerative spondylolisthesis (14.28%, and foraminal stenosis (7.1%. The Oswestry Low-Back Pain Disability Questionnaire and visual analog scale (VAS for pain were recorded preoperatively and at the third and twelfth postoperative months. Results. Both the Oswestry and VAS scores showed significant improvement postoperatively (P<0.05. We observed complications in one patient who had spinal epidural hematoma. Conclusion. We recommend skipping posterior transpedicular dynamic stabilization for surgical treatment of distant segment spinal degenerative disease.

  12. Velocity Building by Reflection Waveform Inversion without Cycle-skipping

    KAUST Repository

    Guo, Q.

    2017-05-26

    Reflection waveform inversion (RWI) provides estimation of low wavenumber model components using reflections generated from a migration/demigration process. The resulting model tends to be a good initial model for FWI. In fact, the optimization images to combine the migration velocity analysis (MVA) objectives (given here by RWI) and the FWI ones. However, RWI may still encounter cycle-skipping at far offsets if the velocity model is highly inaccurate. Similar to MVA, RWI is devoted to focusing reflection data to its true image positions, yet because of the cycle skipping potential we tend to initially use only near offsets. To make the inversion procedure more robust, we introduce the extended image into our RWI. Extending the model perturbations (or image) allows us to better fit the data at larger offsets even with an inaccurate velocity. Thus, we implement a nested approach to optimize the velocity and extended image simultaneously using the objective function of RWI. We slowly reduce the extension, as the image becomes focused, to allow wavepath updates from far offsets to near as a natural progression from long wavelength updates to shorter ones. Applications on synthetic data demonstrate the effectiveness of our method without much additional cost to RWI.

  13. Dynamics of Leading-strand Lesion Skipping by the Replisome

    Science.gov (United States)

    Yeeles, Joseph T.P.; Marians, Kenneth J.

    2013-01-01

    SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

  14. The Detector System for the Stratospheric Kinetic Inductance Polarimeter (SKIP)

    CERN Document Server

    Johnson, B R; Araujo, D; Bradford, K J; Chapman, D; Didier, J; Doyle, S; Eriksen, H K; Flanigan, D; Groppi, C; Hillbrand, S; Jones, G; Limon, M; Mauskopf, P; McCarrick, H; Miller, A; Mroczkowski, T; Reichborn-Kjennerud, B; Smiley, B; Sobrin, J; Wehus, I K; Zmuidzinas, J

    2013-01-01

    We discuss the detector system for the Stratospheric Kinetic Inductance Polarimeter (SKIP). SKIP is a proposed balloon-borne experiment designed to study the cosmic microwave background, the cosmic infrared background and Galactic dust emission by observing 1133 square degrees of sky in the Northern Hemisphere with launches from Kiruna, Sweden. The instrument contains 2317 single-polarization, horn-coupled, aluminum lumped-element kinetic inductance detectors (LEKIDs). The LEKIDs will be maintained at 100 mK with an adiabatic demagnetization refrigerator. The polarimeter operates in two configurations, one sensitive to a spectral band centered on 150 GHz and the other sensitive to 260 and 350 GHz bands. The detector readout system is based on the ROACH-1 board, and the detectors will be biased below 300 MHz. The detector array is fed by an F/2.4 crossed-Dragone telescope with a 500 mm aperture yielding a 15 arcmin FWHM beam at 150 GHz. To minimize detector loading and maximize sensitivity, the entire optical ...

  15. The "alternative" choice of constitutive exons throughout evolution.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    2007-11-01

    Full Text Available Alternative cassette exons are known to originate from two processes-exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 5' splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicing.

  16. Prevention of exercised induced cardiomyopathy following Pip-PMO treatment in dystrophic mdx mice.

    Science.gov (United States)

    Betts, Corinne A; Saleh, Amer F; Carr, Carolyn A; Hammond, Suzan M; Coenen-Stass, Anna M L; Godfrey, Caroline; McClorey, Graham; Varela, Miguel A; Roberts, Thomas C; Clarke, Kieran; Gait, Michael J; Wood, Matthew J A

    2015-03-11

    Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder caused by mutations in the Dmd gene. In addition to skeletal muscle wasting, DMD patients develop cardiomyopathy, which significantly contributes to mortality. Antisense oligonucleotides (AOs) are a promising DMD therapy, restoring functional dystrophin protein by exon skipping. However, a major limitation with current AOs is the absence of dystrophin correction in heart. Pip peptide-AOs demonstrate high activity in cardiac muscle. To determine their therapeutic value, dystrophic mdx mice were subject to forced exercise to model the DMD cardiac phenotype. Repeated peptide-AO treatments resulted in high levels of cardiac dystrophin protein, which prevented the exercised induced progression of cardiomyopathy, normalising heart size as well as stabilising other cardiac parameters. Treated mice also exhibited significantly reduced cardiac fibrosis and improved sarcolemmal integrity. This work demonstrates that high levels of cardiac dystrophin restored by Pip peptide-AOs prevents further deterioration of cardiomyopathy and pathology following exercise in dystrophic DMD mice.

  17. Transposable elements in disease-associated cryptic exons.

    Science.gov (United States)

    Vorechovsky, Igor

    2010-02-01

    Transposable elements (TEs) make up a half of the human genome, but the extent of their contribution to cryptic exon activation that results in genetic disease is unknown. Here, a comprehensive survey of 78 mutation-induced cryptic exons previously identified in 51 disease genes revealed the presence of TEs in 40 cases (51%). Most TE-containing exons were derived from short interspersed nuclear elements (SINEs), with Alus and mammalian interspersed repeats (MIRs) covering >18 and >16% of the exonized sequences, respectively. The majority of SINE-derived cryptic exons had splice sites at the same positions of the Alu/MIR consensus as existing SINE exons and their inclusion in the mRNA was facilitated by phylogenetically conserved changes that improved both traditional and auxiliary splicing signals, thus marking intronic TEs amenable for pathogenic exonization. The overrepresentation of MIRs among TE exons is likely to result from their high average exon inclusion levels, which reflect their strong splice sites, a lack of splicing silencers and a high density of enhancers, particularly (G)AA(G) motifs. These elements were markedly depleted in antisense Alu exons, had the most prominent position on the exon-intron gradient scale and are proposed to promote exon definition through enhanced tertiary RNA interactions involving unpaired (di)adenosines. The identification of common mechanisms by which the most dynamic parts of the genome contribute both to new exon creation and genetic disease will facilitate detection of intronic mutations and the development of computational tools that predict TE hot-spots of cryptic exon activation.

  18. The Dystrophin-Glycoprotein Complex in the Prevention of Muscle Damage

    Directory of Open Access Journals (Sweden)

    Jessica D. Gumerson

    2011-01-01

    Full Text Available Muscular dystrophies are genetically diverse but share common phenotypic features of muscle weakness, degeneration, and progressive decline in muscle function. Previous work has focused on understanding how disruptions in the dystrophin-glycoprotein complex result in muscular dystrophy, supporting a hypothesis that the muscle sarcolemma is fragile and susceptible to contraction-induced injury in multiple forms of dystrophy. Although benign in healthy muscle, contractions in dystrophic muscle may contribute to a higher degree of muscle damage which eventually overwhelms muscle regeneration capacity. While increased susceptibility of muscle to mechanical injury is thought to be an important contributor to disease pathology, it is becoming clear that not all DGC-associated diseases share this supposed hallmark feature. This paper outlines experimental support for a function of the DGC in preventing muscle damage and examines the evidence that supports novel functions for this complex in muscle that when impaired, may contribute to the pathogenesis of muscular dystrophy.

  19. Leptin Level and Skipping Breakfast: The National Health and Nutrition Examination Survey III (NHANES III)

    Science.gov (United States)

    Asao, Keiko; Marekani, Amandine Sambira; VanCleave, Jessica; Rothberg, Amy E.

    2016-01-01

    Skipping breakfast is a common dietary habit considered to be unhealthy. However, the mechanisms underlying skipping breakfast have not been fully explored. Leptin is a hormone that regulates food intake and energy storage and secretes in a diurnal rhythm with lowest levels in the morning. We examined the association between the serum leptin level and skipping breakfast in 5714 adults in the U.S. National Health and Nutrition Examination Survey III, 1988–1994. We defined breakfast as any food or beverage consumed between 5:00 a.m. and 10:00 a.m. using a single 24-h recall. Skipped breakfast was seen in 13.1%. In the logistic regression models with and without adjusting for adiposity and sex, leptin levels were not associated with skipping breakfast. After adjusting for age, race/ethnicity, and time of venipuncture, the association remained insignificant. After further adjusting for potential confounders: physical activity, alcohol intake, smoking and diabetes and after further adjusting for: dietary factors, insulin and glucose levels, there was a 9% and 11%–12%, respectively, statistically significantly higher likelihood of skipping breakfast if the leptin level was more than 50% greater. Further investigation into the biological reasons for skipping breakfast may be useful for promoting healthy lifestyles. PMID:26927164

  20. Lifestyle and socioeconomic correlates of breakfast skipping in Hong Kong primary 4 schoolchildren.

    Science.gov (United States)

    Tin, Sze Pui Pamela; Ho, Sai Yin; Mak, Kwok Hang; Wan, Ka Leung; Lam, Tai Hing

    2011-01-01

    Although breakfast is associated with different benefits, breakfast skipping is increasingly common among children. This study aimed to identify lifestyle and socioeconomic correlates of breakfast skipping in Hong Kong schoolchildren. 68,606 primary 4 participants of the Department of Health Student Health Service in 1998-2000 reported breakfast habit and other lifestyle characteristics using a standardized questionnaire. Height and weight were measured by trained SHS nurses. Socioeconomic data were reported by parents. In cross-sectional analysis, multivariate logistic regression was used to identify lifestyle and socioeconomic correlates of breakfast skipping. 3,598 subjects (5.2%) usually skipped breakfast. Breakfast skipping was associated with being overweight (Odds ratio=1.59, 95% CI: 1.46 to 1.73) and obese (2.06, 1.80 to 2.36), and unhealthy dietary habits including more frequent junk food (1.23, 1.14 to 1.33) but less frequent fruit/vegetable (1.23, 1.13 to 1.34) and milk (1.98, 1.80 to 2.16) intake. Breakfast skippers tended to skip lunch, do less extra-curricular physical activity, watch more television and have less educated parents. Breakfast skipping was significantly related to various health-compromising lifestyle characteristics and lower parental education. Breakfast habit can be a potential lifestyle indicator. Education programmes aimed at specific target groups should encourage regular breakfast consumption. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Leptin Level and Skipping Breakfast: The National Health and Nutrition Examination Survey III (NHANES III).

    Science.gov (United States)

    Asao, Keiko; Marekani, Amandine Sambira; VanCleave, Jessica; Rothberg, Amy E

    2016-02-25

    Skipping breakfast is a common dietary habit considered to be unhealthy. However, the mechanisms underlying skipping breakfast have not been fully explored. Leptin is a hormone that regulates food intake and energy storage and secretes in a diurnal rhythm with lowest levels in the morning. We examined the association between the serum leptin level and skipping breakfast in 5714 adults in the U.S. National Health and Nutrition Examination Survey III, 1988-1994. We defined breakfast as any food or beverage consumed between 5:00 a.m. and 10:00 a.m. using a single 24-h recall. Skipped breakfast was seen in 13.1%. In the logistic regression models with and without adjusting for adiposity and sex, leptin levels were not associated with skipping breakfast. After adjusting for age, race/ethnicity, and time of venipuncture, the association remained insignificant. After further adjusting for potential confounders: physical activity, alcohol intake, smoking and diabetes and after further adjusting for: dietary factors, insulin and glucose levels, there was a 9% and 11%-12%, respectively, statistically significantly higher likelihood of skipping breakfast if the leptin level was more than 50% greater. Further investigation into the biological reasons for skipping breakfast may be useful for promoting healthy lifestyles.

  2. Leptin Level and Skipping Breakfast: The National Health and Nutrition Examination Survey III (NHANES III

    Directory of Open Access Journals (Sweden)

    Keiko Asao

    2016-02-01

    Full Text Available Skipping breakfast is a common dietary habit considered to be unhealthy. However, the mechanisms underlying skipping breakfast have not been fully explored. Leptin is a hormone that regulates food intake and energy storage and secretes in a diurnal rhythm with lowest levels in the morning. We examined the association between the serum leptin level and skipping breakfast in 5714 adults in the U.S. National Health and Nutrition Examination Survey III, 1988–1994. We defined breakfast as any food or beverage consumed between 5:00 a.m. and 10:00 a.m. using a single 24-h recall. Skipped breakfast was seen in 13.1%. In the logistic regression models with and without adjusting for adiposity and sex, leptin levels were not associated with skipping breakfast. After adjusting for age, race/ethnicity, and time of venipuncture, the association remained insignificant. After further adjusting for potential confounders: physical activity, alcohol intake, smoking and diabetes and after further adjusting for: dietary factors, insulin and glucose levels, there was a 9% and 11%–12%, respectively, statistically significantly higher likelihood of skipping breakfast if the leptin level was more than 50% greater. Further investigation into the biological reasons for skipping breakfast may be useful for promoting healthy lifestyles.

  3. Effect of breakfast skipping on diurnal variation of energy metabolism and blood glucose.

    Science.gov (United States)

    Kobayashi, Fumi; Ogata, Hitomi; Omi, Naomi; Nagasaka, Shoichiro; Yamaguchi, Sachiko; Hibi, Masanobu; Tokuyama, Kumpei

    2014-01-01

    Epidemiological studies suggest an association between breakfast skipping and body weight gain, insulin resistance or type 2 diabetes. Time when meal is consumed affects postprandial increase in energy expenditure and blood glucose, and breakfast skipping may reduce 24 h energy expenditure and elevate blood glucose level. The present study evaluated the effect of breakfast skipping on diurnal variation of energy metabolism and blood glucose. The skipped breakfast was compensated by following big meals at lunch and supper. In a randomized repeated-measure design with or without breakfast, eight males stayed twice in a room-size respiratory chamber. Blood glucose was recorded with a continuous glucose monitoring system. Breakfast skipping did not affect 24 h energy expenditure, fat oxidation and thermic effect of food, but increased overall 24 h average of blood glucose (83 ± 3 vs 89 ± 2 mg/dl, P breakfast skipping. These observations suggest that changes in glucose homeostasis precede that of energy balance, in the potential sequence caused by breakfast skipping, if this dietary habit has any effect on energy balance.:

  4. Proteasome inhibitor (MG132 rescues Nav1.5 protein content and the cardiac sodium current in dystrophin-deficient mdx5cv mice

    Directory of Open Access Journals (Sweden)

    Jean-Sebastien eRougier

    2013-03-01

    Full Text Available The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex (DMC at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (INa were both decreased in cardiomyocytes of dystrophin-deficient mdx5cv mice. In this study, wild-type (WT and mdx5cv mice were treated for 7 days with the proteasome inhibitor MG132 (10 µg/Kg/24 h using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and INa but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.

  5. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

    Energy Technology Data Exchange (ETDEWEB)

    Fujimoto, Takahiro; Itoh, Kyoko, E-mail: kxi14@koto.kpu-m.ac.jp; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  6. New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye

    Directory of Open Access Journals (Sweden)

    Rishko Valentyna M

    2011-09-01

    Full Text Available Abstract Background The Dystrophin Glycoprotein Complex (DGC is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys and Dystroglycan (Dg are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Previously we performed an array of genetic screens using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, we have here continued to analyze the DGC modifiers' function in the developing Drosophila brain and eye. Results Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, we have determined the function of previously screened components and their genetic interaction with the DGC in this tissue. Our study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. We determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. Conclusions Our data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton.

  7. The proton pump inhibitor lansoprazole improves the skeletal phenotype in dystrophin deficient mdx mice.

    Directory of Open Access Journals (Sweden)

    Arpana Sali

    Full Text Available BACKGROUND: In Duchenne muscular dystrophy (DMD, loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology. METHODOLOGY/PRINCIPAL FINDINGS: We designed a preclinical trial to investigate the effects of lansoprazole (LANZO administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group: (1 vehicle control; (2 5 mg/kg/day LANZO; (3 5 mg/kg/day prednisolone; and (4 combined treatment of 5 mg/kg/day prednisolone (PRED and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan and functional outcomes (grip strength and Rotarod were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology, in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions. CONCLUSIONS/SIGNIFICANCE: Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings

  8. Associations between perceived friends' support of healthy eating and meal skipping in adolescence.

    Science.gov (United States)

    Rosenrauch, Sharon; Ball, Kylie; Lamb, Karen E

    2017-09-07

    Meal skipping is a relatively common behaviour during adolescence. As peer influence increases during adolescence, friendship groups may play a role in determining eating patterns such as meal skipping. The current study examined cross-sectional and longitudinal associations between perceived friends' support of healthy eating and breakfast and lunch skipping among adolescents. Survey of intrapersonal, social and environmental factors that may influence eating patterns at baseline (2004/05) and follow-up (2006/07). Thirty-seven secondary schools in Victoria, Australia. Sample of 1785 students aged 12-15 years at baseline. Adolescents who reported that their friends sometimes or often ate healthy foods with them were less likely (adjusted OR; 95 % CI) to skip breakfast (sometimes: 0·71; 0·57, 0·90; often: 0·54; 0·38, 0·76) or lunch (sometimes: 0·61; 0·41, 0·89; often: 0·59; 0·37, 0·94) at baseline than those who reported their friends never or rarely displayed this behaviour. Although this variable was associated with lunch skipping at follow-up, there was no evidence of an association with breakfast skipping at follow-up. There was no evidence of an association between perceived encouragement of healthy eating, and an inconsistent relationship between perceived discouragement of junk food consumption, and meal skipping. Friends eating healthy foods together may serve to reduce meal skipping during early adolescence, possibly due to the influence of directly observable behaviour and shared beliefs held by those in the same friendship group. Verbal encouragement or discouragement from friends may be less impactful an influence on meal skipping (than directly observable behaviours) in adolescents.

  9. Possible influences on the expression of X chromosome-linked dystrophin abnormalities by heterozygosity for autosomal recessive Fukuyama congenital muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Beggs, A.H.; Neumann, P.E.; Anderson, M.S.; Kunkel, L.M. (Harvard Medical School, Boston, MA (United States)); Arahata, Kiichi; Arikawa, Eri; Nonaka, Ikuya (National Inst. of Neuroscience, Tokyo (Japan))

    1992-01-15

    Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3,500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, the authors propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1.

  10. Exonic splicing regulatory elements skew synonymous codon usage near intron-exon boundaries in mammals.

    NARCIS (Netherlands)

    Parmley, J.L.; Hurst, L.D.

    2007-01-01

    In mammals there is a bias in amino acid usage near splice sites that is explained, in large part, by the high density of exonic splicing enhancers (ESEs) in these regions. Is there a similar bias for the relative use of synonymous codons, and can any such bias be predicted by their abundance in ESE

  11. Analysis of factors influencing skip lymphatic metastasis in pN2 non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    Gui-Long Li; Yong Zhu; Wei Zheng; Chao-Hui Guo; Chun Chen

    2012-01-01

    Objective:Although many clinical studies on skip lymphatic metastasis in non-small cell lung cancer have been reported,the risk factors for skip lymphatic metastasis are still controversy and debatable.This study investigated,by multivariate logistic regression analysis,the clinical features of skip metastasis to mediastinal lymph nodes (N2) in non-small cell lung cancer (NSCLC) patients.Methods:We collected the clinicopathological data of 256 pN2-NSCLC patients who underwent lobectomy plus systemic lymph node dissection in Fujian Medical University Union Hospital.The cases in the present study were divided into two groups:skip metastasis (N2 skip+) and non-skip metastasis (N2 skip-).A retrospective analysis of clinical pathological features of two groups was performed.To determine an independent factor,multivariate logistic regression analysis was used to identify possible risk factors.Results:A total of 256 pN2-NSCLC patients were recruited.The analysis results showed that gender,pathologic types,surgery,pleural involvement,smoking history,age,tumor stages,and differentiation were not statistical significant factors impacting on skip metastasis in pN2-NSCLC (P>0.05),whereas tumor size was an independent factor for skip metastasis (P=0.02).Conclusions:The rate of skip lymphatic metastasis increases in pN2-NSCLC patients,in accompany with an increased tumor size.

  12. Species-Specific Exon Loss in Human Transcriptomes

    OpenAIRE

    Wang, Jinkai; Lu, Zhi-xiang; Tokheim, Collin J.; Miller, Sara E.; Xing, Yi

    2014-01-01

    Changes in exon–intron structures and splicing patterns represent an important mechanism for the evolution of gene functions and species-specific regulatory networks. Although exon creation is widespread during primate and human evolution and has been studied extensively, much less is known about the scope and potential impact of human-specific exon loss events. Historically, transcriptome data and exon annotations are significantly biased toward humans over nonhuman primates. This ascertainm...

  13. Many U.S. Travelers Skip Measles Shots, Despite Infection Risk

    Science.gov (United States)

    ... news/fullstory_165583.html Many U.S. Travelers Skip Measles Shots, Despite Infection Risk Though the disease was ... eligible Americans traveling abroad don't get a measles vaccine, and a key reason is lack of ...

  14. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy

    OpenAIRE

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of exten...

  15. Exon Deletions of Parkin Gene in Patients with Parkinson Disease

    Institute of Scientific and Technical Information of China (English)

    王涛; 梁直厚; 孙圣刚; 曹学兵; 彭海; 刘红进; 童萼塘

    2004-01-01

    Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.

  16. Characteristics of transposable element exonization within human and mouse.

    Directory of Open Access Journals (Sweden)

    Noa Sela

    Full Text Available Insertion of transposed elements within mammalian genes is thought to be an important contributor to mammalian evolution and speciation. Insertion of transposed elements into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization. Elucidation of the evolutionary constraints that have shaped fixation of transposed elements within human and mouse protein coding genes and subsequent exonization is important for understanding of how the exonization process has affected transcriptome and proteome complexities. Here we show that exonization of transposed elements is biased towards the beginning of the coding sequence in both human and mouse genes. Analysis of single nucleotide polymorphisms (SNPs revealed that exonization of transposed elements can be population-specific, implying that exonizations may enhance divergence and lead to speciation. SNP density analysis revealed differences between Alu and other transposed elements. Finally, we identified cases of primate-specific Alu elements that depend on RNA editing for their exonization. These results shed light on TE fixation and the exonization process within human and mouse genes.

  17. Palladium-catalyzed 1,4-difunctionalization of butadiene to form skipped polyenes.

    Science.gov (United States)

    McCammant, Matthew S; Liao, Longyan; Sigman, Matthew S

    2013-03-20

    A palladium-catalyzed 1,4-addition across the commodity chemical 1,3-butadiene to afford skipped polyene products is reported. Through a palladium σ → π → σ allyl isomerization, two new carbon-carbon bonds are formed with high regioselectivity and trans stereoselectivity of the newly formed alkene. The utility of this method is highlighted by the successful synthesis of the ripostatin A skipped triene core.

  18. Lifestyle and socioeconomic correlates of breakfast skipping in Hong Kong primary 4 schoolchildren

    OpenAIRE

    Tin, SPP; Ho, SY; Mak, KH; Wan, KL; Lam, Th

    2011-01-01

    Objective: Although breakfast is associated with different benefits, breakfast skipping is increasingly common among children. This study aimed to identify lifestyle and socioeconomic correlates of breakfast skipping in Hong Kong schoolchildren. Methods: 68 606 primary 4 participants of the Department of Health Student Health Service in 1998-2000 reported breakfast habit and other lifestyle characteristics using a standardized questionnaire. Height and weight were measured by trained SHS nurs...

  19. The relationship between breakfast skipping, chronotype, and glycemic control in type 2 diabetes.

    Science.gov (United States)

    Reutrakul, Sirimon; Hood, Megan M; Crowley, Stephanie J; Morgan, Mary K; Teodori, Marsha; Knutson, Kristen L

    2014-02-01

    Breakfast skipping is associated with obesity and an increased risk of type 2 diabetes. Later chronotypes, individuals who have a preference for later bed and wake times, often skip breakfast. The aim of the study was to explore the relationships among breakfast skipping, chronotype, and glycemic control in type 2 diabetes patients. We collected sleep timing and 24-h dietary recall from 194 non-shift-working type 2 diabetes patients who were being followed in outpatient clinics. Mid-sleep time on free days (MSF) was used as an indicator of chronotype. Hemoglobin A1C (HbA1C) values were obtained from medical records. Hierarchical linear regression analyses controlling for demographic, sleep, and dietary variables were computed to determine whether breakfast skipping was associated with HbA1C. Additional regression analyses were performed to test if this association was mediated by chronotype. There were 22 participants (11.3%) who self-reported missing breakfast. Breakfast skippers had significantly higher HbA1C levels, higher body mass indices (BMI), and later MSF than breakfast eaters. Breakfast skipping was significantly associated with higher HbA1C values (B = 0.108, p = 0.01), even after adjusting for age, sex, race, BMI, number of diabetes complications, insulin use, depressive symptoms, perceived sleep debt, and percentage of daily caloric intake at dinner. The relationship between breakfast skipping and HbA1C was partially mediated by chronotype. In summary, breakfast skipping is associated with a later chronotype. Later chronotype and breakfast skipping both contribute to poorer glycemic control, as indicated by higher HbA1C levels. Future studies are needed to confirm these findings and determine whether behavioral interventions targeting breakfast eating or sleep timing may improve glycemic control in patients with type 2 diabetes.

  20. Localization and quantitation of the chromosome 6-encoded dystrophin-related protein in normal and pathological human muscle.

    Science.gov (United States)

    Karpati, G; Carpenter, S; Morris, G E; Davies, K E; Guerin, C; Holland, P

    1993-03-01

    A dystrophin-related protein (DRP) encoded by a gene on chromosome 6 was studied in 14 normal and 79 pathological human skeletal muscle biopsies, as well as in cultured myotubes by light microscopic immunocytochemistry and quantitative immunoblots. In normal muscle immunoreactive DRP was present at the postjunctional surface membrane, at the surface of satellite cells, in the walls of blood vessels, in Schwann cells and in perineurium of intramuscular nerves. All of this produced a weak signal on immunoblots. In Duchenne/Becker dystrophy (DMD/BMD) and in polymyositis (PM) or dermatomyositis (DM) DRP was present throughout the extrajunctional surface membrane of extra- and intrafusal muscle fibers, particularly regenerating ones. This produced a 15-17-fold increase of DRP over normal in DMD/BMD and 4-10-fold increase over normal in PM and DM on immunoblots. In other pathological muscles, DRP localization pattern and quantity was about the same as in normals. Dystrophin-related protein was present in about the same amounts and distribution in normal and DMD cultured myoblasts and myotubes. The molecular stimulus for the marked upregulation of DRP in DMD/BMD and in the inflammatory myopathies is not known. In DMD/BMD the diffuse sarcolemmal DRP may partially compensate for dystrophin deficiency.

  1. Muscle dysfunction and structural defects of dystrophin-null sapje mutant zebrafish larvae are rescued by ataluren treatment.

    Science.gov (United States)

    Li, Mei; Andersson-Lendahl, Monika; Sejersen, Thomas; Arner, Anders

    2014-04-01

    Sapje zebrafish carry a mutation in the dystrophin gene, which results in a premature stop codon, and a severe muscle phenotype. They display several of the structural characteristics of Duchenne muscular dystrophy (DMD). Ataluren (PTC124) is proposed to cause readthrough of premature stop codons and has been introduced as a potential treatment of genetic disorders. Clinical trials in DMD have shown promise, although with complex dose dependency. We have established physiology techniques, enabling high resolution of contractile function in skeletal muscle of zebrafish larvae. We aimed to provide a mechanical analysis of sapje larval muscle and examine effects of ataluren. Homozygous 5 d postfertilization (dpf) sapje larvae exhibited structural defects with 50% decrease in active tension. Ataluren (0.1-1 μM, 3-5 dpf) improved contractile function (~60% improvement of force at 0.5 μM) and dystrophin expression. Controls were not affected. Higher doses (5 μM, 35 μM) impaired contractile function, an effect also observed in controls, suggesting unspecific negative effects at high concentrations. In summary, Sapje larvae exhibit impaired contractile performance and provide a relevant DMD model for functional studies. Ataluren significantly improves skeletal muscle function in the sapje larvae, most likely reflecting an observed increase in dystrophin expression. The bell-shaped dose dependence in sapje resembles that previously reported in clinical DMD studies.

  2. An alpha-catulin homologue controls neuromuscular function through localization of the dystrophin complex and BK channels in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Linu S Abraham

    2010-08-01

    Full Text Available The large conductance, voltage- and calcium-dependent potassium (BK channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 Mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.

  3. Skipping of the very-high-frequency structural particle de () in Chinese reading.

    Science.gov (United States)

    Zang, Chuanli; Zhang, Manman; Bai, Xuejun; Yan, Guoli; Angele, Bernhard; Liversedge, Simon P

    2017-01-16

    How do readers decide whether to skip or fixate a word? Angele and Rayner [2013. Processing the in the parafovea: Are articles skipped automatically? Journal of Experimental Psychology: Learning, Memory, and Cognition, 39, 649-662] showed that English readers base skipping decisions on the parafoveal information available, but not the sentential context. Due to the increased visual density of the language, Chinese readers may be able to process a parafoveal word and integrate it with the sentence context to a greater extent than English readers. Consequently, influences on skipping decisions in Chinese may differ from those in English. In a boundary paradigm experiment, participants read sentences containing a single-character target verb (e.g., meaning get) whose preview was manipulated in three conditions: identity preview; a preview consisting of the syntactically anomalous high-frequency structural particle de (), or a pseudocharacter preview. The results showed that Chinese readers were more likely to skip the target when the preview was de than in either of the other conditions, suggesting that de-skipping is triggered by the parafoveal preview of a highly frequent particle word rather than on the likelihood of the upcoming word given the sentential context..

  4. Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product.

    Science.gov (United States)

    Datson, N A; van de Vosse, E; Dauwerse, H G; Bout, M; van Ommen, G J; den Dunnen, J T

    1996-03-15

    To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple exons of the Duchenne Muscular Dystrophy gene. All exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single exon trap product rather than small single exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of exon trap material.

  5. Aberrant location of inhibitory synaptic marker proteins in the hippocampus of dystrophin-deficient mice: implications for cognitive impairment in duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Elżbieta Krasowska

    Full Text Available Duchenne muscular dystrophy (DMD is a neuromuscular disease that arises from mutations in the dystrophin-encoding gene. Apart from muscle pathology, cognitive impairment, primarily of developmental origin, is also a significant component of the disorder. Convergent lines of evidence point to an important role for dystrophin in regulating the molecular machinery of central synapses. The clustering of neurotransmitter receptors at inhibitory synapses, thus impacting on synaptic transmission, is of particular significance. However, less is known about the role of dystrophin in influencing the precise expression patterns of proteins located within the pre- and postsynaptic elements of inhibitory synapses. To this end, we exploited molecular markers of inhibitory synapses, interneurons and dystrophin-deficient mouse models to explore the role of dystrophin in determining the stereotypical patterning of inhibitory connectivity within the cellular networks of the hippocampus CA1 region. In tissue from wild-type (WT mice, immunoreactivity of neuroligin2 (NL2, an adhesion molecule expressed exclusively in postsynaptic elements of inhibitory synapses, and the vesicular GABA transporter (VGAT, a marker of GABAergic presynaptic elements, were predictably enriched in strata pyramidale and lacunosum moleculare. In acute contrast, NL2 and VGAT immunoreactivity was relatively evenly distributed across all CA1 layers in dystrophin-deficient mice. Similar changes were evident with the cannabinoid receptor 1, vesicular glutamate transporter 3, parvalbumin, somatostatin and the GABAA receptor alpha1 subunit. The data show that in the absence of dystrophin, there is a rearrangement of the molecular machinery, which underlies the precise spatio-temporal pattern of GABAergic synaptic transmission within the CA1 sub-field of the hippocampus.

  6. The effect of high- and low-frequency previews and sentential fit on word skipping during reading

    Science.gov (United States)

    Angele, Bernhard; Laishley, Abby; Rayner, Keith; Liversedge, Simon P.

    2014-01-01

    In a previous gaze-contingent boundary experiment, Angele and Rayner (2012) found that readers are likely to skip a word that appears to be the definite article the even when syntactic constraints do not allow for articles to occur in that position. In the present study, we investigated whether the word frequency of the preview of a three-letter target word influences a reader’s decision to fixate or skip that word. We found that the word frequency rather than the felicitousness (syntactic fit) of the preview affected how often the upcoming word was skipped. These results indicate that visual information about the upcoming word trumps information from the sentence context when it comes to making a skipping decision. Skipping parafoveal instances of the therefore may simply be an extreme case of skipping high-frequency words. PMID:24707791

  7. Dystrophin-deficient dogs with reduced myostatin have unequal muscle growth and greater joint contractures.

    Science.gov (United States)

    Kornegay, Joe N; Bogan, Daniel J; Bogan, Janet R; Dow, Jennifer L; Wang, Jiahui; Fan, Zheng; Liu, Naili; Warsing, Leigh C; Grange, Robert W; Ahn, Mihye; Balog-Alvarez, Cynthia J; Cotten, Steven W; Willis, Monte S; Brinkmeyer-Langford, Candice; Zhu, Hongtu; Palandra, Joe; Morris, Carl A; Styner, Martin A; Wagner, Kathryn R

    2016-01-01

    Myostatin (Mstn) is a negative regulator of muscle growth whose inhibition promotes muscle growth and regeneration. Dystrophin-deficient mdx mice in which myostatin is knocked out or inhibited postnatally have a less severe phenotype with greater total mass and strength and less fibrosis and fatty replacement of muscles than mdx mice with wild-type myostatin expression. Dogs with golden retriever muscular dystrophy (GRMD) have previously been noted to have increased muscle mass and reduced fibrosis after systemic postnatal myostatin inhibition. Based partly on these results, myostatin inhibitors are in development for use in human muscular dystrophies. However, persisting concerns regarding the effects of long-term and profound myostatin inhibition will not be easily or imminently answered in clinical trials. To address these concerns, we developed a canine (GRippet) model by crossbreeding dystrophin-deficient GRMD dogs with Mstn-heterozygous (Mstn (+/-)) whippets. A total of four GRippets (dystrophic and Mstn (+/-)), three GRMD (dystrophic and Mstn wild-type) dogs, and three non-dystrophic controls from two litters were evaluated. Myostatin messenger ribonucleic acid (mRNA) and protein levels were downregulated in both GRMD and GRippet dogs. GRippets had more severe postural changes and larger (more restricted) maximal joint flexion angles, apparently due to further exaggeration of disproportionate effects on muscle size. Flexors such as the cranial sartorius were more hypertrophied on magnetic resonance imaging (MRI) in the GRippets, while extensors, including the quadriceps femoris, underwent greater atrophy. Myostatin protein levels negatively correlated with relative cranial sartorius muscle cross-sectional area on MRI, supporting a role in disproportionate muscle size. Activin receptor type IIB (ActRIIB) expression was higher in dystrophic versus control dogs, consistent with physiologic feedback between myostatin and ActRIIB. However, there was no

  8. Deletion of the Chd6 exon 12 affects motor coordination.

    Science.gov (United States)

    Lathrop, Melissa J; Chakrabarti, Lisa; Eng, Jeremiah; Rhodes, C Harker; Lutz, Thomas; Nieto, Amelia; Liggitt, H Denny; Warner, Sandra; Fields, Jennifer; Stöger, Reinhard; Fiering, Steven

    2010-04-01

    Members of the CHD protein family play key roles in gene regulation through ATP-dependent chromatin remodeling. This is facilitated by chromodomains that bind histone tails, and by the SWI2/SNF2-like ATPase/helicase domain that remodels chromatin by moving histones. Chd6 is ubiquitously expressed in both mouse and human, with the highest levels of expression in the brain. The Chd6 gene contains 37 exons, of which exons 12-19 encode the highly conserved ATPase domain. To determine the biological role of Chd6, we generated mouse lines with a deletion of exon 12. Chd6 without exon 12 is expressed at normal levels in mice, and Chd6 Exon 12 -/- mice are viable, fertile, and exhibit no obvious morphological or pathological phenotype. Chd6 Exon 12 -/- mice lack coordination as revealed by sensorimotor analysis. Further behavioral testing revealed that the coordination impairment was not due to muscle weakness or bradykinesia. Histological analysis of brain morphology revealed no differences between Chd6 Exon 12 -/- mice and wild-type (WT) controls. The location of CHD6 on human chromosome 20q12 is overlapped by the linkage map regions of several human ataxias, including autosomal recessive infantile cerebellar ataxia (SCAR6), a nonprogressive cerebrospinal ataxia. The genomic location, expression pattern, and ataxic phenotype of Chd6 Exon 12 -/- mice indicate that mutations within CHD6 may be responsible for one of these ataxias.

  9. Longitudinal ambulatory measurements of gait abnormality in dystrophin-deficient dogs

    Directory of Open Access Journals (Sweden)

    Voit Thomas

    2011-04-01

    Full Text Available Abstract Background This study aimed to measure the gait abnormalities in GRMD (Golden retriever muscular dystrophy dogs during growth and disease progression using an ambulatory gait analyzer (3D-accelerometers as a possible tool to assess the effects of a therapeutic intervention. Methods Six healthy and twelve GRMD dogs were evaluated twice monthly, from the age of two to nine months. The evolution of each gait variable previously shown to be modified in control and dystrophin-deficient adults was assessed using two-ways variance analysis (age, clinical status with repeated measurements. A principal component analysis (PCA was applied to perfect multivariate data interpretation. Results Speed, stride length, total power and force significantly already decreased (p Conclusion The gait variables measured by the accelerometers were sensitive to early detect and follow the gait disorders and mirrored the heterogeneity of clinical presentations, giving sense to monitor gait in GRMD dogs during progression of the disease and pre-clinical therapeutic trials.

  10. Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.

    Directory of Open Access Journals (Sweden)

    Mariya M Kucherenko

    Full Text Available The Dystroglycan-Dystrophin (Dg-Dys complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-beta and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought.

  11. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

    Science.gov (United States)

    Fujimoto, Takahiro; Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  12. Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model

    Directory of Open Access Journals (Sweden)

    Jahnke Vanessa E

    2012-08-01

    Full Text Available Abstract Background Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice. Methods Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR, separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s' effects. Results We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage. Conclusions The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice.

  13. Detection of deletion in the dystrophin gene of a patient with quadriceps myopathy.

    Directory of Open Access Journals (Sweden)

    Kumari D

    2000-01-01

    Full Text Available A 43 year old male presented with slowly progressive weakness of limbs and hypertrophy of triceps, brachioradialis and calf muscles for four years. There was thinning of quadriceps muscles in both thighs. Histological study was compatible with Becker muscular dystrophy (BMD. Genomic DNA analysis showed a deletion of the Hind III fragments, spanning exons 45-47. A junction fragment of 11.0 kb was observed along with a deletion of a 3.4 kb PstI fragment containing exon 51 in the patient, and in one of his two sisters. The clinical and laboratory characteristics in this patient are in keeping with what has been described ′quadriceps myopathy′ and fall within the phenotypic variants of BMD as has been shown by others.

  14. Relationships between bullying victimization psychological distress and breakfast skipping among boys and girls.

    Science.gov (United States)

    Sampasa-Kanyinga, Hugues; Willmore, Jacqueline

    2015-06-01

    The purpose of this study was to further explore the association between bullying victimization and breakfast skipping in children and adolescents. Compared to the previous study, we have used a larger and representative sample of middle and high school students, examined the effect of gender, different forms (physical, verbal, theft/vandalism and cyber) and severity of bullying on breakfast eating behaviour. Data from students (2286 boys and 2859 girls) aged 11 to 19 years (mean ± SD age: 14.6 ± 1.9 years) from the 2013 Ontario Student Drug Use and Health Survey (OSDUHS) were analysed using self-reports of being bullied, diet, psychological distress, demographics, socio-economic status, weight status, and substance use. Results revealed greater odds of breakfast skipping in girl victims of physical, verbal, and cyber bullying, and in boy victims of verbal and cyber bullying. There was a dose-response relationship between experience of both school and cyber bullying victimization and breakfast skipping behaviour for both genders. Mediation analysis indicated that psychological distress fully mediated the relationship between both verbal and physical bullying victimization and breakfast skipping in girls, and partially mediated the relationship between verbal bullying victimization and breakfast skipping in boys. Psychological distress also partially mediated the link between cyber bullying victimization and breakfast skipping in both boys and girls. These results corroborate previous findings on the association between bullying victimization and breakfast skipping in children and adolescents. The strong and consistent associations with different forms of bullying victimization, the dose-response relationship, and the mediating role of psychological distress suggest a causal relationship. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Simultaneous Pathoproteomic Evaluation of the Dystrophin-Glycoprotein Complex and Secondary Changes in the mdx-4cv Mouse Model of Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Sandra Murphy

    2015-06-01

    Full Text Available In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue.

  16. Skip the trip: air travelers' behavioral responses to pandemic influenza.

    Directory of Open Access Journals (Sweden)

    Eli P Fenichel

    Full Text Available Theory suggests that human behavior has implications for disease spread. We examine the hypothesis that individuals engage in voluntary defensive behavior during an epidemic. We estimate the number of passengers missing previously purchased flights as a function of concern for swine flu or A/H1N1 influenza using 1.7 million detailed flight records, Google Trends, and the World Health Organization's FluNet data. We estimate that concern over "swine flu," as measured by Google Trends, accounted for 0.34% of missed flights during the epidemic. The Google Trends data correlates strongly with media attention, but poorly (at times negatively with reported cases in FluNet. Passengers show no response to reported cases. Passengers skipping their purchased trips forwent at least $50 M in travel related benefits. Responding to actual cases would have cut this estimate in half. Thus, people appear to respond to an epidemic by voluntarily engaging in self-protection behavior, but this behavior may not be responsive to objective measures of risk. Clearer risk communication could substantially reduce epidemic costs. People undertaking costly risk reduction behavior, for example, forgoing nonrefundable flights, suggests they may also make less costly behavior adjustments to avoid infection. Accounting for defensive behaviors may be important for forecasting epidemics, but linking behavior with epidemics likely requires consideration of risk communication.

  17. Why do some adult birds skip breeding? A hormonal investigation in a long-lived bird.

    Science.gov (United States)

    Goutte, Aurélie; Kriloff, Marion; Weimerskirch, Henri; Chastel, Olivier

    2011-10-23

    Skipping reproduction is often observed in long-lived organisms, but proximate mechanisms remain poorly understood. Since young and/or very old snow petrels (Pagodroma nivea) commonly skip breeding, we tested whether they are physiologically able to breed during the pre-laying stage. To do so, we measured the ability of known-age (11-45 years old) petrels to release luteinizing hormone (LH, a crucial driver for breeding), by injecting exogenous gonadotropin-releasing hormone (GnRH). Although young petrels exhibited low baseline LH levels, they were able to elevate LH levels after a GnRH challenge. Moreover, young and very old petrels showed a stronger decrease in LH levels after the 10 min post-GnRH injection compared with middle-aged petrels. Birds that skipped breeding were as able as breeders to release LH after a GnRH challenge, indicating that they had functional pituitaries. However, the decision to skip reproduction was linked to a strong LH decrease after the 10 min post-GnRH injection. Our result suggests that the youngest and the oldest petrels fail to maintain elevated baseline LH levels, thereby do not initiate reproductive activities. Skipping reproduction in long-lived birds probably results from age-related changes in the dynamics of the hypothalamic-pituitary-gonadal (HPG) axis function.

  18. Health, behavioral, cognitive, and social correlates of breakfast skipping among women living in socioeconomically disadvantaged neighborhoods.

    Science.gov (United States)

    Smith, Kylie J; McNaughton, Sarah A; Cleland, Verity J; Crawford, David; Ball, Kylie

    2013-11-01

    Breakfast skipping is a potentially modifiable behavior that has negative effects on health and is socioeconomically patterned. This study aimed to examine the intrapersonal (health, behavioral, and cognitive) and social factors associated with breakfast skipping. Nonpregnant women (n = 4123) aged 18-45 y from socioeconomically disadvantaged neighborhoods throughout Victoria, Australia, completed a postal questionnaire. Sociodemographic characteristics, diet, physical activity, sedentary behaviors, and cognitive and social factors were assessed by self-report. Breakfast skipping was defined in 2 ways: 1) "rarely/never" eating breakfast (n = 498) and 2) eating breakfast ≤2 d/wk (includes those who rarely/never ate breakfast; n = 865). Poisson regression was used to calculate prevalence ratios and linear trends, adjusting for covariates. The P values for linear trends are reported below. Compared with breakfast consumers, women who reported rarely/never eating breakfast tended to have poorer self-rated health (P-trend breakfast skipping was defined as eating breakfast ≤2 d/wk, additional associations were found for having lower leisure-time physical activity (P-trend = 0.012) and less self-efficacy for eating a healthy diet (P-trend breakfast skipping among women living in socioeconomically disadvantaged areas. Acknowledging the cross-sectional design and need for causal confirmation, programs that aim to promote breakfast consumption in this population group should consider targeting family-related barriers to healthy eating and nutrition knowledge.

  19. Sequence Analysis of Hoxc8 Exon-1 and Exon-2 of Multi-Vertebrae Mongolia Sheep%多脊椎蒙古羊Hoxc8 exon-1和exon-2的序列分析

    Institute of Scientific and Technical Information of China (English)

    陈琦; 赵静; 张立岭; 马月辉

    2011-01-01

    参考牛的Hoxc8基因序列设计引物,扩增正常蒙古羊(胸椎数13)和多脊椎蒙古羊(胸椎数14)Hoxc8的exon-1和exon-2基因,对得到的序列进行生物信息学分析。结果表明,经序列比对二者的DNA序列,除两侧个别碱基有差异外,中间序列完全一致。蒙古羊Hoxc8的exon-1和exon-2序列分别与其他物种进行同源性比对,蒙古羊Hoxc8 exon-1与人、小鼠、大鼠、犬的同源性达到96%以上,与斑马鱼的同源性为75.8%;exon-2与大猩猩、犬、人、小鼠、大鼠的同源性达到91%以上,与斑马鱼的同源性%In our study,according to the Hoxc8 sequence of cow,the specific primers were designed,and the sequences of Hoxc8 exon-1(432 bp)and exon-2(273 bp)of normal and multi-thoracic vertebrae mongolia sheep were obtained(Genebank accession number: EU817489 and FJ905472).Alignment results of them indicated that the sequences were conformity except a little difference in two sides of sequences.Hoxc8 exon-1 and exon-2 were aligned with other species and the results showed that compared with other mammals(human,dog,mouse,rat and chimpanzee),the homology were above 96%(exon-1) and 91%(exon-2);compared with zebra fish,the homology were 75.8% and 74%.

  20. A Two-amino Acid Mutation Encountered in Duchenne Muscular Dystrophy Decreases Stability of the Rod Domain 23 (R23) Spectrin-like Repeat of Dystrophin.

    Science.gov (United States)

    Legardinier, Sébastien; Legrand, Baptiste; Raguénès-Nicol, Céline; Bondon, Arnaud; Hardy, Serge; Tascon, Christophe; Le Rumeur, Elisabeth; Hubert, Jean-François

    2009-03-27

    Lack of functional dystrophin causes severe Duchenne muscular dystrophy. The subsarcolemmal location of dystrophin, as well as its association with both cytoskeleton and membrane, suggests a role in the mechanical regulation of muscular membrane stress. In particular, phenotype rescue in a Duchenne muscular dystrophy mice model has shown that some parts of the central rod domain of dystrophin, constituted by 24 spectrin-like repeats, are essential. In this study, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in muscle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on three-dimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability. We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle.

  1. Nestin expression in end-stage disease in dystrophin-deficient heart: implications for regeneration from endogenous cardiac stem cells.

    Science.gov (United States)

    Berry, Suzanne E; Andruszkiewicz, Peter; Chun, Ju Lan; Hong, Jun

    2013-11-01

    Nestin(+) cardiac stem cells differentiate into striated cells following myocardial infarct. Transplantation of exogenous stem cells into myocardium of a murine model for Duchenne muscular dystrophy (DMD) increased proliferation of endogenous nestin(+) stem cells and resulted in the appearance of nestin(+) striated cells. This correlated with, and may be responsible for, prevention of dilated cardiomyopathy. We examined nestin(+) stem cells in the myocardium of dystrophin/utrophin-deficient (mdx/utrn(-/-)) mice, a model for DMD. We found that 92% of nestin(+) interstitial cells expressed Flk-1, a marker present on cardiac progenitor cells that differentiate into the cardiac lineage, and that a subset expressed Sca-1, present on adult cardiac cells that become cardiomyocytes. Nestin(+) interstitial cells maintained expression of Flk-1 but lost Sca-1 expression with age and were present in lower numbers in dystrophin-deficient heart than in wild-type heart. Unexpectedly, large clusters of nestin(+) striated cells ranging in size from 20 to 250 cells and extending up to 500 μm were present in mdx/utrn(-/-) heart near the end stage of disease. These cells were also present in dystrophin-deficient mdx/utrn(+/-) and mdx heart but not wild-type heart. Nestin(+) striated cells expressed cardiac troponin I, desmin, and Connexin 43 and correlated with proinflammatory CD68(+) macrophages. Elongated nestin(+) interstitial cells with striations were observed that did not express Flk-1 or the late cardiac marker cardiac troponin I but strongly expressed the early cardiac marker desmin. Nestin was also detected in endothelial and smooth muscle cells. These data indicate that new cardiomyocytes form in dystrophic heart, and nestin(+) interstitial cells may generate them in addition to other cells of the cardiac lineage.

  2. Ultrastructural changes in the interstitial cells of Cajal and gastric dysrhythmias in mice lacking full-length dystrophin (mdx mice).

    Science.gov (United States)

    Vannucchi, Maria-Giuliana; Zizzo, Maria-Grazia; Zardo, Claudio; Pieri, Laura; Serio, Rosa; Mulè, Flavia; Faussone-Pellegrini, Maria-Simonetta

    2004-05-01

    At least two populations of c-kit positive interstitial cells of Cajal (ICC) lie in the gastric wall, one located at the myenteric plexus level has a pace-making function and the other located intramuscularly is intermediary in the neurotransmission and regenerates the slow waves. Both of these ICC sub-types express full-length dystrophin. Mdx mice, an animal model lacking in full-length dystrophin and used to study Duchenne muscular dystrophy (DMD), show gastric dismotilities. The aim of the present study was to verify in mdx mice whether: (i) gastric ICC undergo morphological changes, through immunohistochemical and ultrastructural analyses; and (ii) there are alterations in the electrical activity, using intracellular recording technique. In control mice, ICC sub-types showed heterogeneous ultrastructural features, either intramuscularly or at the myenteric plexus level. In mdx mice, all of the ICC sub-types underwent important changes: coated vesicles were significantly more numerous and caveolae significantly fewer than in control; moreover, cytoskeleton and smooth endoplasmic reticulum were reduced and mitochondria enlarged. c-Kit-positivity and integrity of the ICC networks were maintained. In the circular muscle of normal mice slow waves, which consisted of initial and secondary components, occurred with a regular frequency. In mdx mice, slow waves occurred in a highly dysrhythmic fashion and they lacked a secondary component. We conclude that the lack of the full-length dystrophin is associated with ultrastructural modifications of gastric ICC, most of which can be interpreted as signs of new membrane formation and altered Ca(2+) handling, and with defective generation and regeneration of slow wave activity.

  3. Dystrophin is required for the normal function of the cardio-protective K(ATP channel in cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Laura Graciotti

    Full Text Available Duchenne and Becker muscular dystrophy patients often develop a cardiomyopathy for which the pathogenesis is still unknown. We have employed the murine animal model of Duchenne muscular dystrophy (mdx, which develops a cardiomyopathy that includes some characteristics of the human disease, to study the molecular basis of this pathology. Here we show that the mdx mouse heart has defects consistent with alteration in compounds that regulate energy homeostasis including a marked decrease in creatine-phosphate (PC. In addition, the mdx heart is more susceptible to anoxia than controls. Since the cardio-protective ATP sensitive potassium channel (K(ATP complex and PC have been shown to interact we investigated whether deficits in PC levels correlate with other molecular events including K(ATP ion channel complex presence, its functionality and interaction with dystrophin. We found that this channel complex is present in the dystrophic cardiac cell membrane but its ability to sense a drop in the intracellular ATP concentration and consequently open is compromised by the absence of dystrophin. We further demonstrate that the creatine kinase muscle isoform (CKm is displaced from the plasma membrane of the mdx cardiac cells. Considering that CKm is a determinant of K(ATP channel complex function we hypothesize that dystrophin acts as a scaffolding protein organizing the K(ATP channel complex and the enzymes necessary for its correct functioning. Therefore, the lack of proper functioning of the cardio-protective K(ATP system in the mdx cardiomyocytes may be part of the mechanism contributing to development of cardiac disease in dystrophic patients.

  4. Distribution of dystrophin- and utrophin-associated protein complexes (DAPC/UAPC) in human hematopoietic stem/progenitor cells.

    Science.gov (United States)

    Teniente-De Alba, Carmen; Martínez-Vieyra, Ivette; Vivanco-Calixto, Raúl; Galván, Iván J; Cisneros, Bulmaro; Cerecedo, Doris

    2011-10-01

    Hematopoietic stem cells (HSC) are defined by their cardinal properties, such as sustained proliferation, multilineage differentiation, and self-renewal, which give rise to a hierarchy of progenitor populations with more restricted potential lineage, ultimately leading to the production of all types of mature blood cells. HSC are anchored by cell adhesion molecules to their specific microenvironment, thus regulating their cell cycle, while cell migration is essentially required for seeding the HSC of the fetal bone marrow (BM) during development as well as in adult BM homeostasis. The dystrophin-associated protein complex (DAPC) is a large group of membrane-associated proteins linking the cytoskeleton to the extracellular matrix and exhibiting scaffolding, adhesion, and signaling roles in muscle and non-muscle cells including mature blood cells. Because adhesion and migration are mechanisms that influence the fate of the HSC, we explored the presence and the feasible role of DAPC. In this study, we characterized the pattern expression by immunoblot technique and, by confocal microscopy analysis, the cellular distribution of dystrophin and utrophin gene products, and the dystrophin-associated proteins (α-, β-dystroglycan, α-syntrophin, α-dystrobrevin) in relation to actin filaments in freshly isolated CD34+ cells from umbilical cord blood. Immunoprecipitation assays demonstrated the presence of Dp71d/Dp71Δ110m ∼DAPC and Up400/Up140∼DAPC. The subcellular distribution of the two DAPC in actin-based structures suggests their dynamic participation in adhesion and cell migration. In addition, the particular protein pattern expression found in hematopoietic stem/progenitor cells might be indicative of their feasible participation during differentiation.

  5. Potential Association between Breakfast Skipping and Concomitant Late-Night-Dinner Eating with Metabolic Syndrome and Proteinuria in the Japanese Population

    OpenAIRE

    Ayano Kutsuma; Kei Nakajima; Kaname Suwa

    2014-01-01

    Skipping breakfast is considered to be an unhealthy eating habit linked to predispositions to obesity and type 2 diabetes. Because eating dinner late at night can elicit subsequent breakfast skipping, we investigated if skipping breakfast concomitant with late-night-dinner eating (LNDE) was associated with metabolic syndrome (MetS) and proteinuria in the general Japanese population. We examined self-reported habitual breakfast skipping and LNDE, MetS (modified ATP-III criteria), and proteinur...

  6. Adolescents' unhealthy eating habits are associated with meal skipping.

    Science.gov (United States)

    Rodrigues, Paulo Rogério Melo; Luiz, Ronir Raggio; Monteiro, Luana Silva; Ferreira, Márcia Gonçalves; Gonçalves-Silva, Regina Maria Veras; Pereira, Rosangela Alves

    2017-10-01

    Meal consumption and diet quality are important for healthy development during adolescence. The aim of this study was to determine the association between meal habits and diet quality in Brazilian adolescents. A school-based, cross-sectional study was conducted in 2008 with a probabilistic sample of adolescents ages 14 to 19 y (N = 1139) from high schools in central-western Brazil. Consumption of breakfast, morning snack, lunch, afternoon snack, and dinner was assessed to evaluate adolescents' meal profile. The Brazilian Healthy Eating Index-Revised (BHEI-R) was calculated to evaluate diet quality. The association between meal profile and BHEI-R (global estimates and components) was assessed using multivariate linear regression models. Diet was characterized by unhealthy eating: a low consumption of fruits, vegetables, and milk/dairy, and a high consumption of fats and sodium. An unsatisfactory meal profile was observed in 14% of adolescents, whereas daily consumption of breakfast, lunch, and dinner was reported by 47%, 78%, and 52% of adolescents, respectively. Meal profile was positively associated with diet quality. Daily consumption of breakfast was associated with higher BHEI-R scores, lower sodium intake, and greater consumption of fruits and milk/dairy. Daily consumption of lunch was associated with greater consumption of vegetables and "meats, eggs, and legumes," whereas consumption of dinner was associated with an increased consumption of "whole fruits." This study showed a parallelism between daily consumption of meals with healthier eating and greater adherence to traditional Brazilian food habits. Skipping meals was associated with a low-quality diet, especially concerning to the low consumption of fruits and vegetables and a high intake of sodium and calories from solid fats, added sugars, and alcoholic beverages. Therefore, the adoption of regular meal habits may help adolescents improve their diet quality. Copyright © 2017 Elsevier Inc. All

  7. Skipping breakfast and overweight in 2-and 5-year-old Dutch children-the GECKO Drenthe cohort

    NARCIS (Netherlands)

    Kupers, L. K.; de Pijper, J. J.; Sauer, P. J. J.; Stolk, R. P.; Corpeleijn, E.

    2014-01-01

    Skipping breakfast is associated with higher BMI in children aged 5 years and older. However, not much is known about this association in younger children. In the Dutch GECKO Drenthe birth cohort we examined the association between breakfast skipping and objectively measured overweight at the age of

  8. Skipping breakfast and overweight in 2-and 5-year-old Dutch children-the GECKO Drenthe cohort

    NARCIS (Netherlands)

    Kupers, L. K.; de Pijper, J. J.; Sauer, P. J. J.; Stolk, R. P.; Corpeleijn, E.

    Skipping breakfast is associated with higher BMI in children aged 5 years and older. However, not much is known about this association in younger children. In the Dutch GECKO Drenthe birth cohort we examined the association between breakfast skipping and objectively measured overweight at the age of

  9. Forward recursion for Markov decision processes with skip-free-to-the-right transitions - Part II : Non-standard applications

    NARCIS (Netherlands)

    Wijngaard, J.; Stidham, S.

    2000-01-01

    This paper is the sequel to Wijngaard and Stidham (1986). The topic is a countable state, average reward semi-Markov decision process with a transition mechanism that is skip-free to the right. The applications are controlled GI/M/1 queues. Skip-free to the right means that state n cannot be reached

  10. Grade Skipping from the Perspective of Teachers in Germany: The Links between Teachers' Decisions, Acceptance, and Perceived Knowledge

    Science.gov (United States)

    Westphal, Andrea; Vock, Miriam; Stubbe, Tobias

    2017-01-01

    The present study explored teachers' perspectives on one specific type of acceleration, namely, grade skipping. In addition, we investigated the extent to which teachers' beliefs about students' academic, motivational, and social development after grade skipping may explain teachers' acceptance of this accelerative strategy. Moreover, we examined…

  11. 76 FR 70340 - Generation-Skipping Transfers (GST) Section 6011 Regulations and Amendments to the Section 6112...

    Science.gov (United States)

    2011-11-14

    ... Internal Revenue Service 26 CFR Parts 26 and 301 RIN 1545-BG89 Generation-Skipping Transfers (GST) Section 6011 Regulations and Amendments to the Section 6112 Regulations AGENCY: Internal Revenue Service (IRS... generation-skipping transfer tax under section 6011 of the Internal Revenue Code (Code), conforming...

  12. Disruption of action potential and calcium signaling properties in malformed myofibers from dystrophin-deficient mice.

    Science.gov (United States)

    Hernández-Ochoa, Erick O; Pratt, Stephen J P; Garcia-Pelagio, Karla P; Schneider, Martin F; Lovering, Richard M

    2015-04-01

    Duchenne muscular dystrophy (DMD), the most common and severe muscular dystrophy, is caused by the absence of dystrophin. Muscle weakness and fragility (i.e., increased susceptibility to damage) are presumably due to structural instability of the myofiber cytoskeleton, but recent studies suggest that the increased presence of malformed/branched myofibers in dystrophic muscle may also play a role. We have previously studied myofiber morphology in healthy wild-type (WT) and dystrophic (MDX) skeletal muscle. Here, we examined myofiber excitability using high-speed confocal microscopy and the voltage-sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS) to assess the action potential (AP) properties. We also examined AP-induced Ca(2+) transients using high-speed confocal microscopy with rhod-2, and assessed sarcolemma fragility using elastimetry. AP recordings showed an increased width and time to peak in malformed MDX myofibers compared to normal myofibers from both WT and MDX, but no significant change in AP amplitude. Malformed MDX myofibers also exhibited reduced AP-induced Ca(2+) transients, with a further Ca(2+) transient reduction in the branches of malformed MDX myofibers. Mechanical studies indicated an increased sarcolemma deformability and instability in malformed MDX myofibers. The data suggest that malformed myofibers are functionally different from myofibers with normal morphology. The differences seen in AP properties and Ca(2+) signals suggest changes in excitability and remodeling of the global Ca(2+) signal, both of which could underlie reported weakness in dystrophic muscle. The biomechanical changes in the sarcolemma support the notion that malformed myofibers are more susceptible to damage. The high prevalence of malformed myofibers in dystrophic muscle may contribute to the progressive strength loss and fragility seen in dystrophic muscles. © 2015 The Authors. Physiological Reports published by Wiley

  13. Differential expression of myosin heavy chain isoforms in the masticatory muscles of dystrophin-deficient mice.

    Science.gov (United States)

    Spassov, Alexander; Gredes, Tomasz; Gedrange, Tomasz; Lucke, Silke; Morgenstern, Sven; Pavlovic, Dragan; Kunert-Keil, Christiane

    2011-12-01

    The dystrophin-deficient mouse (mdx) is a homologue animal model of Duchenne muscular dystrophy (DMD) and is characterized by slowly progressive muscle weakness accompanied by changes in myosin heavy chain (MyHC) composition. It is likely that the masticatory muscles undergo similar changes. The aim of this study was to examine the masticatory muscles (masseter, temporal, tongue, and soleus) of 100-day-old mdx and control mice (n = 8-10), and the fibre type distribution (by immunohistochemistry) as well as the expression of the corresponding MyHC messenger RNA (mRNA) (protein and mRNA expression, using Western blot or quantitative real-time polymerase chain reaction (RT-PCR)). Immunohistochemistry and western blot analysis revealed that the masticatory muscles in the control and mdx mice consisted mainly of type 2 fibres, whereas soleus muscle consisted of both type 1 and 2 fibres. In the masseter muscle, the mRNA in mdx mice was not different from that found in the controls. However, the mRNA content of the MyHC-2b isoform in mdx mice was lower in comparison with the controls in the temporal muscle [11.9 versus 36.9 per cent; P muscle (65.7 versus 73.8 per cent; P muscle was lower than in the controls (25.9 versus 30.8 per cent; P muscles of mdx mice may lead to changed fibre type composition. The different MyHC gene expression in mdx mice masticatory muscles may be seen as an adaptive mechanism to muscular dystrophy.

  14. An Adaptive Frame Skipping and VOP Interpolation Algorithm for Video Object Segmentation

    Institute of Scientific and Technical Information of China (English)

    YANGGaobo; ZHANGZhaoyang

    2004-01-01

    Video object segmentation is a key step for the successful use of MPEG-4. However, most of the current available segmentation algorithms are still far away from real-time performance. In order to improve the processing speed, an adaptive frame skipping and VOP interpolation algorithm is proposed in this paper. It adaptively determines the number of skipped frames based on the rigidity and motion complexity of video object. To interpolate the VOPs for skipped frames, a hi-directional projection scheme is adopted. Its principle is to perform a classification of those regions obtained by spatial segmentation for every frame in the sequence. It is valid for both rigid object and non-rigid object and can get good localization of object boundaries. Experimental results show that the proposed approach can improve the processing speed greatly while maintaining visually pleasant results.

  15. Skip-webs: Efficient distributed data structures for multi-dimensional data sets

    DEFF Research Database (Denmark)

    Arge, Lars; Eppstein, David; Goodrich, Michael T.

    2005-01-01

    We present a framework for designing efficient distributed data structures for multi-dimensional data. Our structures, which we call skip-webs, extend and improve previous randomized distributed data structures, including skipnets and skip graphs. Our framework applies to a general class of data...... querying scenarios, which include linear (one-dimensional) data, such as sorted sets, as well as multi-dimensional data, such as d-dimensional octrees and digital tries of character strings defined over a fixed alphabet. We show how to perform a query over such a set of n items spread among n hosts using O......, and O(log n / log log n) messages for one-dimensional data. Finally, we show how to apply a blocking strategy to skip-webs to further improve message complexity for one-dimensional data when hosts can store more data....

  16. Family factors for child meal skipping in low-income families in Korea.

    Science.gov (United States)

    Bae, Hwa-ok; Kim, Meesook; Hong, Soon-Myung

    2010-04-01

    The present study proposed to examine whether family factors are associated with child meal skipping in Korea. Family factors were divided into risk factors and protective factors on conceptual and theoretical bases. The sample was obtained from the Survey of Meal Service for Poor Children conducted by the Korea Institute for Health and Social Affairs in 2007. A final sample was composed of 944 children in low-income families who are served by the public meal program. Child meal skipping was positively associated with risk factors and negatively associated with protective factors, as hypothesized. Single-father family, middle or small urban area, presence of caretaker after school, health level of caretaker, caretaker's concern about child's diet, and degree of family cohesion significantly predicted child meal skipping. The authors suggest a few implications for practice based on the study findings.

  17. Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product.

    OpenAIRE

    Datson, N.A.; Vosse, E van de; Dauwerse, H.G.; Bout, M; van Ommen, G J; J T den Dunnen

    1996-01-01

    To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is de...

  18. Does Skipping Breakfast and Being Overweight Influence Academic Achievement Among Korean Adolescents?

    Science.gov (United States)

    Kang, Yang Wha; Park, Jong-Hyock

    2016-08-01

    Health status and health behaviors are associated with academic achievement in children and adolescents. The purpose of this study was to investigate whether skipping breakfast and being overweight are related to academic achievement of Korean adolescents. Cross-sectional data on a sample of 1,652 high-school seniors (942 males and 710 females) drawn from the 2004 Korea Education Employment Panel were analyzed. A higher proportion of males (15.3%) than females (6.1%) was overweight (p < 0.001); 37% of males and 41% of females reported skipping breakfast. Overall test scores were significantly higher for females than males (p < 0.05), and in language and foreign language subjects. However, both males and females who reported skipping breakfast had significantly lower scores in language, mathematics, and foreign language than those who did not report skipping breakfast. Overweight males had a lower probability than normal-weight males of having the highest language scores (OR = 0.52, p < 0.05), but there was no difference among females. Females who skipped breakfast had a lower probability of having the highest scores in language (OR = 0.41, p < 0.05), mathematics (OR = 0.24, p < 0.01), or foreign language (OR = 0.18, p < 0.01), while males had a lower probability of having the highest scores in language only (OR = 0.46, p < 0.05). Skipping breakfast and being overweight are associated with poor academic achievement in Korean adolescents. Eating breakfast and weight control is being discussed as the overlooked factors that may influence better academic achievement.

  19. Factors associated with skipping breakfast among Inner Mongolia Medical students in China

    Directory of Open Access Journals (Sweden)

    Sun Juan

    2013-01-01

    Full Text Available Abstract Background Few studies on the breakfast consumption habits of medical students in China have been carried out. The aim of the present study was to determine the prevalence of skipping breakfast and factors associated with skipping breakfast among medical students in Inner Mongolia of China, and to assist in the design of interventions to improve breakfast consumption habits of medical college students in this region. Methods From December 2010 to January 2011 a cross-sectional survey was conducted among medical students in the Inner Mongolia Medical College using a self-administered questionnaire. The prevalence of skipping breakfast in relation to lifestyle habits was described and factors associated with breakfast consumption were identified using multiple logistic regression analysis. Results The overall prevalence of skipping breakfast was 41.7% and 23.5% for males and females, respectively. The Faculty of Medicine Information Management had the highest breakfast skipping prevalence. Logistic regression models found that the main factors associated with breakfast consumption habits among medical students were gender, class years of education, monthly expenses, faculty, appetite, sleeping quality, and the learning process; monthly expenses, sleeping quality, and the learning process showed a dose-dependent relationship. Conclusions Breakfast consumption was associated with many factors, most importantly monthly expenses, sleeping quality and the learning process. The prevalence of skipping breakfast is significantly higher compared recently reported figures for medical students in western countries and other areas of China. Improvement of breakfast education should be considered for students in which higher monthly expenses, poor sleeping quality, or a laborious learning process have been identified.

  20. Differentiated evolutionary rates in alternative exons and the implications for splicing regulation

    Directory of Open Access Journals (Sweden)

    Eyras Eduardo

    2006-06-01

    Full Text Available Abstract Background Alternatively spliced exons play an important role in the diversification of gene function in most metazoans and are highly regulated by conserved motifs in exons and introns. Two contradicting properties have been associated to evolutionary conserved alternative exons: higher sequence conservation and higher rate of non-synonymous substitutions, relative to constitutive exons. In order to clarify this issue, we have performed an analysis of the evolution of alternative and constitutive exons, using a large set of protein coding exons conserved between human and mouse and taking into account the conservation of the transcript exonic structure. Further, we have also defined a measure of the variation of the arrangement of exonic splicing enhancers (ESE-conservation score to study the evolution of splicing regulatory sequences. We have used this measure to correlate the changes in the arrangement of ESEs with the divergence of exon and intron sequences. Results We find evidence for a relation between the lack of conservation of the exonic structure and the weakening of the sequence evolutionary constraints in alternative and constitutive exons. Exons in transcripts with non-conserved exonic structures have higher synonymous (dS and non-synonymous (dN substitution rates than exons in conserved structures. Moreover, alternative exons in transcripts with non-conserved exonic structure are the least constrained in sequence evolution, and at high EST-inclusion levels they are found to be very similar to constitutive exons, whereas alternative exons in transcripts with conserved exonic structure have a dS significantly lower than average at all EST-inclusion levels. We also find higher conservation in the arrangement of ESEs in constitutive exons compared to alternative ones. Additionally, the sequence conservation at flanking introns remains constant for constitutive exons at all ESE-conservation values, but increases for

  1. Origin of introns by 'intronization' of exonic sequences

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David;

    2008-01-01

    The mechanisms of spliceosomal intron creation have proved elusive. Here we describe a new mechanism: the recruitment of internal exonic sequences ('intronization') in Caenorhabditis species. The numbers of intronization events and introns gained by other mechanisms are similar, suggesting...

  2. Widespread evolutionary conservation of alternatively spliced exons in caenorhabditis

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Penny, David

    2007-01-01

    Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern...

  3. Lampe1: an ENU-germline mutation causing spontaneous hepatosteatosis identified through targeted exon-enrichment and next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Rachel Sheridan

    Full Text Available Using a small scale ENU mutagenesis approach we identified a recessive germline mutant, designated Lampe1 that exhibited growth retardation and spontaneous hepatosteatosis. Low resolution mapping based on 20 intercrossed Lampe1 mice revealed linkage to a ∼14 Mb interval on the distal site of chromosome 11 containing a total of 285 genes. Exons and 50 bp flanking sequences within the critical region were enriched with sequence capture microarrays and subsequently analyzed by next-generation sequencing. Using this approach 98.1 percent of the targeted DNA was covered with a depth of 10 or more reads per nucleotide and 3 homozygote mutations were identified. Two mutations represented intronic nucleotide changes whereas one mutation affected a splice donor site in intron 11-12 of Palmitoyl Acetyl-coenzyme A oxygenase-1 (Acox1, causing skipping of exon 12. Phenotyping of Acox1(Lampe1 mutants revealed a progression from hepatosteatosis to steatohepatitis, and ultimately hepatocellular carcinoma. The current approach provides a highly efficient and affordable method to identify causative mutations induced by ENU mutagenesis and animal models relevant to human pathology.

  4. Media use as a reason for meal skipping and fast eating in secondary school children.

    Science.gov (United States)

    Van den Bulck, J; Eggermont, S

    2006-04-01

    This study examined self-reported meal skipping and eating faster than usual with the goal of watching television or playing computer games. Respondents reported their media use and indicated how often they skipped a meal to watch a favourite television programme or to play a computer game, and how often they ate faster than usual in order to watch television or play a computer game. Respondents were 2546 adolescents of 13 (first year of secondary school) and 16 years (fourth year of secondary school) of age. About one respondent in 10 skipped at least one meal every week for either television viewing or computer game playing. Weekly meal skipping for television viewing occurs more regularly in boys and first-year students, but particularly in teenagers who view 5 h or more daily (15% of the sample). The category of teenagers who play computer games four times a week or more (25.3% of the sample) is at increased risk of meal skipping; those who play more than four times a week are 10 times more likely weekly to skip a meal. A quarter of the adolescents eat faster at least once a week to be able to watch television or play a computer game. Regardless of gender and school year, teenagers' risk of eating faster progressively increases with their use of the media. Those who watch 4 h or more daily are about seven times more likely to skip a meal for television and those who play computer games at least four times a week are nine times more likely weekly to skip a meal. Unhealthy eating habits can be a side effect of heavy or excessive media use. Teenagers' use of television or game computers during nonworking or out-of-school hours partly displaces the amount of time that needs to be spent at meals. Practitioners and educators may try to encourage or restore a pattern of healthful meal consumption habits by reducing the amount of media use, and by supporting parental rule-making regarding children's eating habits and media use.

  5. Fault Tolerant Variable Block Carry Skip Logic (VBCSL) using Parity Preserving Reversible Gates

    CERN Document Server

    Islam, Md Saiful; Begum, Zerina; Hafiz, Mohd Zulfiquar

    2010-01-01

    Reversible logic design has become one of the promising research directions in low power dissipating circuit design in the past few years and has found its application in low power CMOS design, digital signal processing and nanotechnology. This paper presents the efficient design approaches of fault tolerant carry skip adders (FTCSAs) and compares those designs with the existing ones. Variable block carry skip logic (VBCSL) using the fault tolerant full adders (FTFAs) has also been developed. The designs are minimized in terms of hardware complexity, gate count, constant inputs and garbage outputs. Besides of it, technology independent evaluation of the proposed designs clearly demonstrates its superiority with the existing counterparts.

  6. Tdp-43 cryptic exons are highly variable between cell types.

    Science.gov (United States)

    Jeong, Yun Ha; Ling, Jonathan P; Lin, Sophie Z; Donde, Aneesh N; Braunstein, Kerstin E; Majounie, Elisa; Traynor, Bryan J; LaClair, Katherine D; Lloyd, Thomas E; Wong, Philip C

    2017-02-02

    TDP-43 proteinopathy is a prominent pathological feature that occurs in a number of human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and inclusion body myositis (IBM). Our recent finding that TDP-43 represses nonconserved cryptic exons led us to ask whether cell type-specific cryptic exons could exist to impact unique molecular pathways in brain or muscle. In the present work, we investigated TDP-43's function in various mouse tissues to model disease pathogenesis. We generated mice to conditionally delete TDP-43 in excitatory neurons or skeletal myocytes and identified the cell type-specific cryptic exons associated with TDP-43 loss of function. Comparative analysis of nonconserved cryptic exons in various mouse cell types revealed that only some cryptic exons were common amongst stem cells, neurons, and myocytes; the majority of these nonconserved cryptic exons were cell type-specific. Our results suggest that in human disease, TDP-43 loss of function may impair cell type-specific pathways.

  7. Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Lewis

    2010-01-01

    Full Text Available Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

  8. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

    Science.gov (United States)

    Li, Mei; Arner, Anders

    2015-01-01

    Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf) of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf) was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM). This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively.

  9. Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

    Science.gov (United States)

    Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

    2000-01-01

    Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

  10. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

    Directory of Open Access Journals (Sweden)

    Mei Li

    Full Text Available Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM. This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively.

  11. 免疫组织化学dystrophin染色诊断Duchenne型肌营养不良症的研究%Diagnosis of Duchenne muscular dystrophy through dystrophin expression detection by immunohistochemistry

    Institute of Scientific and Technical Information of China (English)

    刘鹏; 沈定国

    2008-01-01

    目的 探讨Duchenne型肌营养不良症(DMD)肌萎缩蛋白(dystrophin)表达规律和临床意义.方法 收集我院7例DMD患者作为试验组,7例非DMD患者为对照组.使用抗dystrophin杆状结构域单抗、免疫组织化学染色,观察肌膜dystrophin表达.结果 7例DMD患者肌细胞膜dystrophin阴性,7例非DMD患者dystrophin染色阳性.结论 证实DMD患者肌细胞膜dystrophin表达阴性,揭示dystrophin缺失是其发病机制,可以作为确诊DMD手段,对临床诊断DMD有实际意义.%Objective To study dystrophin expression in Duchenne muscular dystrophy (DMD) and non-DMD patients. Methods With immunohistochemistry method, using monoclonal antibody of dystrophin, expression of dystrophin was analyzed in 7 DMD patients (experimental group)and 7 non-DMD patients (control group). Results In 7 non-DMD patients, uniform and continuous dystrophin expression was found along the sarolemma, while not in 7 DMD patients. Conclusions Dystrophin expression in myocyte membrane is negative in DMD patients, which indicates that dystrophin loss may be involved in the pathogenesis of DMD. It can be used as a "gold standard" in diagnosing DMD.

  12. Intron phase correlations and the evolution of the intron/exon structure of genes.

    Science.gov (United States)

    Long, M; Rosenberg, C; Gilbert, W

    1995-01-01

    Two issues in the evolution of the intron/exon structure of genes are the role of exon shuffling and the origin of introns. Using a large data base of eukaryotic intron-containing genes, we have found that there are correlations between intron phases leading to an excess of symmetric exons and symmetric exon sets. We interpret these excesses as manifestations of exon shuffling and make a conservative estimate that at least 19% of the exons in the data base were involved in exon shuffling, suggesting an important role for exon shuffling in evolution. Furthermore, these excesses of symmetric exons appear also in those regions of eukaryotic genes that are homologous to prokaryotic genes: the ancient conserved regions. This last fact cannot be explained in terms of the insertional theory of introns but rather supports the concept that some of the introns were ancient, the exon theory of genes. PMID:8618928

  13. Comparison of Mutation Profiles in the Duchenne Muscular Dystrophy Gene among Populations: Implications for Potential Molecular Therapies

    Directory of Open Access Journals (Sweden)

    Luz Berenice López-Hernández

    2015-03-01

    Full Text Available Novel therapeutic approaches are emerging to restore dystrophin function in Duchenne Muscular Dystrophy (DMD, a severe neuromuscular disease characterized by progressive muscle wasting and weakness. Some of the molecular therapies, such as exon skipping, stop codon read-through and internal ribosome entry site-mediated translation rely on the type and location of mutations. Hence, their potential applicability worldwide depends on mutation frequencies within populations. In view of this, we compared the mutation profiles of the populations represented in the DMD Leiden Open-source Variation Database with original data from Mexican patients (n = 162 with clinical diagnosis of the disease. Our data confirm that applicability of exon 51 is high in most populations, but also show that differences in theoretical applicability of exon skipping may exist among populations; Mexico has the highest frequency of potential candidates for the skipping of exons 44 and 46, which is different from other populations (p < 0.001. To our knowledge, this is the first comprehensive comparison of theoretical applicability of exon skipping targets among specific populations.

  14. Comparison of mutation profiles in the Duchenne muscular dystrophy gene among populations: implications for potential molecular therapies.

    Science.gov (United States)

    López-Hernández, Luz Berenice; Gómez-Díaz, Benjamín; Luna-Angulo, Alexandra Berenice; Anaya-Segura, Mónica; Bunyan, David John; Zúñiga-Guzman, Carolina; Escobar-Cedillo, Rosa Elena; Roque-Ramírez, Bladimir; Ruano-Calderón, Luis Angel; Rangel-Villalobos, Héctor; López-Hernández, Julia Angélica; Estrada-Mena, Francisco Javier; García, Silvia; Coral-Vázquez, Ramón Mauricio

    2015-03-09

    Novel therapeutic approaches are emerging to restore dystrophin function in Duchenne Muscular Dystrophy (DMD), a severe neuromuscular disease characterized by progressive muscle wasting and weakness. Some of the molecular therapies, such as exon skipping, stop codon read-through and internal ribosome entry site-mediated translation rely on the type and location of mutations. Hence, their potential applicability worldwide depends on mutation frequencies within populations. In view of this, we compared the mutation profiles of the populations represented in the DMD Leiden Open-source Variation Database with original data from Mexican patients (n = 162) with clinical diagnosis of the disease. Our data confirm that applicability of exon 51 is high in most populations, but also show that differences in theoretical applicability of exon skipping may exist among populations; Mexico has the highest frequency of potential candidates for the skipping of exons 44 and 46, which is different from other populations (p < 0.001). To our knowledge, this is the first comprehensive comparison of theoretical applicability of exon skipping targets among specific populations.

  15. Fetal microchimeric cells in a fetus-treats-its-mother paradigm do not contribute to dystrophin production in serially parous mdx females.

    Science.gov (United States)

    Seppanen, Elke Jane; Hodgson, Samantha Susan; Khosrotehrani, Kiarash; Bou-Gharios, George; Fisk, Nicholas M

    2012-10-10

    Throughout every pregnancy, genetically distinct fetal microchimeric stem/progenitor cells (FMCs) engraft in the mother, persist long after delivery, and may home to damaged maternal tissues. Phenotypically normal fetal lymphoid progenitors have been described to develop in immunodeficient mothers in a fetus-treats-its-mother paradigm. Since stem cells contribute to muscle repair, we assessed this paradigm in the mdx mouse model of Duchenne muscular dystrophy. mdx females were bred serially to either ROSAeGFP males or mdx males to obtain postpartum microchimeras that received either wild-type FMCs or dystrophin-deficient FMCs through serial gestations. To enhance regeneration, notexin was injected into the tibialis anterior of postpartum mice. FMCs were detected by qPCR at a higher frequency in injected compared to noninjected side muscle (P=0.02). However, the number of dystrophin-positive fibers was similar in mothers delivering wild-type compared to mdx pups. In addition, there was no correlation between FMC detection and percentage dystrophin, and no GFP+ve FMCs were identified that expressed dystrophin. In 10/11 animals, GFP+ve FMCs were detected by immunohistochemistry, of which 60% expressed CD45 with 96% outside the basal lamina defining myofiber contours. Finally we confirmed lack of FMC contribution to statellite cells in postpartum mdx females mated with Myf5-LacZ males. We conclude that the FMC contribution to regenerating muscles is insufficient to have a functional impact.

  16. Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

    Directory of Open Access Journals (Sweden)

    Sandra Murphy

    2015-09-01

    Full Text Available The full-length dystrophin protein isoform of 427 kDa (Dp427, the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context.

  17. Gentamicin treatment in exercised mdx mice: Identification of dystrophin-sensitive pathways and evaluation of efficacy in work-loaded dystrophic muscle.

    Science.gov (United States)

    De Luca, Annamaria; Nico, Beatrice; Rolland, Jean-François; Cozzoli, Anna; Burdi, Rosa; Mangieri, Domenica; Giannuzzi, Viviana; Liantonio, Antonella; Cippone, Valentina; De Bellis, Michela; Nicchia, Grazia Paola; Camerino, Giulia Maria; Frigeri, Antonio; Svelto, Maria; Camerino, Diana Conte

    2008-11-01

    Aminoglycosides force read through of premature stop codon mutations and introduce new mutation-specific gene-corrective strategies in Duchenne muscular dystrophy. A chronic treatment with gentamicin (32 mg/kg/daily i.p., 8-12 weeks) was performed in exercised mdx mice with the dual aim to clarify the dependence on dystrophin of the functional, biochemical and histological alterations present in dystrophic muscle and to verify the long term efficiency of small molecule gene-corrective strategies in work-loaded dystrophic muscle. The treatment counteracted the exercise-induced impairment of in vivo forelimb strength after 6-8 weeks. We observed an increase in dystrophin expression level in all the fibers, although lower than that observed in normal fibers, and found a concomitant recovery of aquaporin-4 at sarcolemma. A significant reduction in centronucleated fibers, in the area of necrosis and in the percentage of nuclear factor-kB-positive nuclei was observed in gastrocnemious muscle of treated animals. Plasma creatine kinase was reduced by 70%. Ex vivo, gentamicin restored membrane ionic conductance in mdx diaphragm and limb muscle fibers. No effects were observed on the altered calcium homeostasis and sarcolemmal calcium permeability, detected by electrophysiological and microspectrofluorimetric approaches. Thus, the maintenance of a partial level of dystrophin is sufficient to reinforce sarcolemmal stability, reducing leakiness, inflammation and fiber damage, while correction of altered calcium homeostasis needs greater expression of dystrophin or direct interventions on the channels involved.

  18. More deletions in the 5{prime} region than in the central region of the dystrophin gene were identified among Filipino Duchenne and Becker muscular dystrophy patients

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-11-06

    This report describes mutations in the dystrophin gene and the frequency of these mutations in Filipino pedigrees with Duchenne and Becker muscular dystrophy (DMD/BMD). The findings suggest the presence of genetic variability among DMD/BMD patients in different populations. 13 refs., 1 tab.

  19. Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy.

    Science.gov (United States)

    Allen, David G; Whitehead, Nicholas P; Froehner, Stanley C

    2016-01-01

    Dystrophin is a long rod-shaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC components cause the muscular dystrophies, a group of inherited diseases in which repeated bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The normal function of dystrophin is poorly defined. In its absence a complex series of changes occur with multiple muscle proteins showing reduced or increased expression or being modified in various ways. In this review, we will consider the various proteins whose expression and function is changed in muscular dystrophies, focusing on Ca(2+)-permeable channels, nitric oxide synthase, NADPH oxidase, and caveolins. Excessive Ca(2+) entry, increased membrane permeability, disordered caveolar function, and increased levels of reactive oxygen species are early changes in the disease, and the hypotheses for these phenomena will be critically considered. The aim of the review is to define the early damage pathways in muscular dystrophy which might be appropriate targets for therapy designed to minimize the muscle degeneration and slow the progression of the disease.

  20. Potential Association between Breakfast Skipping and Concomitant Late-Night-Dinner Eating with Metabolic Syndrome and Proteinuria in the Japanese Population

    Directory of Open Access Journals (Sweden)

    Ayano Kutsuma

    2014-01-01

    Full Text Available Skipping breakfast is considered to be an unhealthy eating habit linked to predispositions to obesity and type 2 diabetes. Because eating dinner late at night can elicit subsequent breakfast skipping, we investigated if skipping breakfast concomitant with late-night-dinner eating (LNDE was associated with metabolic syndrome (MetS and proteinuria in the general Japanese population. We examined self-reported habitual breakfast skipping and LNDE, MetS (modified ATP-III criteria, and proteinuria in a cross-sectional study of 60,800 Japanese adults aged 20–75 years. A total of 14,068 subjects (23.1% skipped breakfast, of whom approximately half (52.8% skipped breakfast alone (without LNDE. The percentages of subjects who skipped breakfast showed a J-shaped relationship with body mass index (BMI. Multivariate logistic regression analysis showed that skipping breakfast concomitant with LNDE (n = 6,645 was significantly associated with MetS and proteinuria, even after adjusting for relevant confounders (odds ratio (95% CI, 1.17 (1.08–1.28, P=0.0003, and 1.37 (1.24–1.52, P<0.0001, resp.. Skipping breakfast alone and LNDE alone were not associated with MetS and proteinuria, respectively. In conclusion, habitual breakfast skipping concomitant with LNDE may represent poorer eating behavior than skipping breakfast alone, associated with MetS, asymptomatic proteinuria, obesity, and low body weight in the general Japanese population.

  1. Potential Association between Breakfast Skipping and Concomitant Late-Night-Dinner Eating with Metabolic Syndrome and Proteinuria in the Japanese Population.

    Science.gov (United States)

    Kutsuma, Ayano; Nakajima, Kei; Suwa, Kaname

    2014-01-01

    Skipping breakfast is considered to be an unhealthy eating habit linked to predispositions to obesity and type 2 diabetes. Because eating dinner late at night can elicit subsequent breakfast skipping, we investigated if skipping breakfast concomitant with late-night-dinner eating (LNDE) was associated with metabolic syndrome (MetS) and proteinuria in the general Japanese population. We examined self-reported habitual breakfast skipping and LNDE, MetS (modified ATP-III criteria), and proteinuria in a cross-sectional study of 60,800 Japanese adults aged 20-75 years. A total of 14,068 subjects (23.1%) skipped breakfast, of whom approximately half (52.8%) skipped breakfast alone (without LNDE). The percentages of subjects who skipped breakfast showed a J-shaped relationship with body mass index (BMI). Multivariate logistic regression analysis showed that skipping breakfast concomitant with LNDE (n = 6,645) was significantly associated with MetS and proteinuria, even after adjusting for relevant confounders (odds ratio (95% CI), 1.17 (1.08-1.28), P = 0.0003, and 1.37 (1.24-1.52), P Skipping breakfast alone and LNDE alone were not associated with MetS and proteinuria, respectively. In conclusion, habitual breakfast skipping concomitant with LNDE may represent poorer eating behavior than skipping breakfast alone, associated with MetS, asymptomatic proteinuria, obesity, and low body weight in the general Japanese population.

  2. strong>TAT> gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping

    DEFF Research Database (Denmark)

    Maydan, G; Andresen, Brage Storstein; Madsen, Pia Pinholt

    2006-01-01

    Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner-Hanhart syndrome). Parents may...

  3. Effects of social determinants on food choice and skipping meals among Turkish adolescents.

    Science.gov (United States)

    Soyer, Meral Turk; Ergin, Isil; Gursoy, Safak Taner

    2008-01-01

    To present data that contributes to understanding factors that influence food choice and skipping meals in adolescents. A cross sectional study is carried in selected high schools in Bornova. Study sample compromises of 527 students chosen randomly by class from a population of 2410 first year in high school students. Self-administered questionnaires containing sociodemographic determinants, self reported weight and height, food choices and meal patterns were used. A psychosocial factor that affects almost all of the students is the "taste and sensory perception of food". The second noticable factor is the "health and nutritious value of food". The time conserved and the convenience in the preparation of food is one of the lifestyle factors that affect more than half of the students. The cost of the food was also found to have an effect. Among the third group of factors categorized as "media", the leading factor is advertisement, effective in one third of the students. Among boys and girls, there was no statistical difference in the type of meal skipped. Living in Izmir for more than 10 years compared to less than ten years, being in a nuclear family to extended family, and belonging to the "owner" social class to "wage laborer" class also do not statistically differ with regard to skipping meals. However, the mother's and father's education level and having a working mother are associated with skipping meals. These results provide important evidence to support opportunities to positively influence the adoption of healthful eating.

  4. Meal-Skipping Behaviors and Body Fat in 6-Year-Old Children

    NARCIS (Netherlands)

    A.I. Wijtzes (Anne); W. Jansen (Wilma); S.H. Bouthoorn (Selma); F.J. van Lenthe (Frank); O.H. Franco (Oscar); A. Hofman (Albert); V.W.V. Jaddoe (Vincent W. V.); H. Raat (Hein)

    2016-01-01

    textabstractObjective To assess the prospective associations of breakfast, lunch, and dinner skipping at age 4 years with body fat (ie, percent fat mass, body mass index [BMI], and weight status) at age 6 years. Study design Data were analyzed from 5913 children participating in the Generation R Stu

  5. Skipping breakfast is associated with lower physical activity energy expenditure in young healthy women

    Science.gov (United States)

    Objective: According to recent NHANES data, the prevalence of breakfast consumption has decreased in adults from 89% to 82% between 1971 and 2002. Skipping breakfast has been negatively correlated with physical activity but positively correlated with body weight and risk factors associated with obes...

  6. Breakfast-skipping in children and young adolescents in the Netherlands

    NARCIS (Netherlands)

    Brugman, E.; Meulmeester, J.F.; Spee-Wekke, A. van der; Verloove-Vanhorick, S.P.

    1998-01-01

    Background: The objective of this study was to provide national figures on the prevalence of breakfast-skipping and the association with sociodemographic variables in 4-15 year old children. Methods: Data of 4,377 children were collected. A food questionnaire (24 h recall) was completed by the paren

  7. Breakfast skipping is associated with cyberbullying and school bullying victimization. A school-based cross-sectional study.

    Science.gov (United States)

    Sampasa-Kanyinga, Hugues; Roumeliotis, Paul; Farrow, Claire V; Shi, Yuanfeng F

    2014-08-01

    Breakfast skipping is a health concern that has well-known negative consequences physically and psychologically. It is therefore important to understand why children skip breakfast. The purpose of this study was to establish whether the experience of bullying and cyberbullying impacts upon breakfast skipping and to further evaluate whether the inability for youths to cope with bullying victimization affects their mental health (depression), and in turn predicts breakfast skipping. Data were obtained from the Eastern Ontario 2011 Youth Risk Behaviour Survey, a cross-sectional regional school-based survey of middle and high school students (11-20 years old) across the five counties of Eastern Ontario, Canada (N = 3035). Self-reported data about children's experiences of bullying victimization, breakfast eating habits, socio-economical status, depression, and other risk behaviours were analysed. Approximately half of the participants (50.4%) reported not eating breakfast on a regular basis: 26.3% and 24.1% reported often (usually eat breakfast three times or more per week) and frequent (usually eat breakfast twice a week or less) breakfast skipping behaviour, respectively. Victims of both cyberbullying and school bullying presented greater likelihood of often (adjusted relative risk ratio (RR) = 1.55; 95% confidence interval (CI) = 1.17-2.06) and frequent (RR = 1.97; 95% CI = 1.28-3.03) breakfast skipping. Mediation analysis further showed that depression fully mediated the relationship between school bullying victimization and frequent breakfast skipping. Moreover, depression partially mediated the associations between both cyberbullying and school bullying with frequent breakfast skipping. These findings highlight the potential interrelationships between cyberbullying, school bullying and depression in predicting unhealthy breakfast skipping behaviour in children.

  8. The combined unhealthy behaviors of breakfast skipping and smoking are associated with the prevalence of diabetes mellitus.

    Science.gov (United States)

    Nishiyama, Midori; Muto, Takashi; Minakawa, Toshihiro; Shibata, Toshie

    2009-08-01

    Skipping breakfast has been considered a representative unhealthy behavior, but there is little information about the combined effects of breakfast skipping and other unhealthy health habits, especially smoking. First this cross-sectional study investigated unhealthy behaviors among breakfast skippers, and then examined the impact of the combined association of skipping breakfast and smoking on health. A total of 1,200 adults living in one Japanese community were sent questionnaires to elicit data on age, gender, breakfast-eating frequency, and other lifestyle habits. A total 603 of people returned their questionnaires (response rate: 50.3%), and 493 (230 men and 263 women) questionnaires were considered appropriate for analysis. Smoking rate in men (mean age, 53.7 years) and women (mean age, 50.4 years) was 41.3%, and 9.5%, respectively. Skipping breakfast was more prevalent in people under age 50 years (p breakfast skipping (3.10, 95%CI 1.50-6.39). Other factors included, age younger than 50 years (3.04, 95%CI 1.31-7.06) and poor sleeping quality (2.06, 95%CI 1.00-4.25). After examining the combined impact of skipping breakfast and smoking, the highest odds ratio for a diagnosis of diabetes mellitus was found among those who smoked and skipped breakfast (4.68, 95% CI: 1.46-15.05). Moreover, skipping breakfast among non-smokers showed a high association with perceived stress (2.83, 95% CI: 1.05-7.61). In conclusion, the combined unhealthy behaviors of skipping breakfast and smoking are associated with the prevalence of diabetes mellitus.

  9. Distribution of components of basal lamina and dystrophin-dystroglycan complex in the rat pineal gland: differences from the brain tissue and between the subdivisions of the gland.

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    Bagyura, Zsolt; Pócsai, Károly; Kálmán, Mihály

    2010-01-01

    The pineal gland is an evagination of the brain tissue, a circumventricular neuroendocrine organ. Our immunohistochemical study investigates basal lamina components (laminin, agrin, perlecan, fibronectin), their receptor, the dystrophin-dystroglycan complex (beta-dystroglycan, dystrophin utrophin), aquaporins (-4,-9) and cellular markers (S100, neurofilament, GFAP, glutamine synthetase) in the adult rat corpus pineale. The aim was to compare the immunohistochemical features of the cerebral and pineal vessels and their environment, and to compare their features in the distal and proximal subdivisions of the so-called 'superficial pineal gland'. In contrast to the cerebral vessels, pineal vessels proved to be immunonegative to alpha1-dystrobrevin, but immunoreactive to laminin. An inner, dense, and an outer, loose layer of laminin as two basal laminae were present. The gap between them contained agrin and perlecan. Basal lamina components enmeshed the pinealocytes, too. Components of dystrophin-dystroglycan complex were also distributed along the vessels. Dystrophin, utrophin and agrin gave a 'patchy' distribution rather than a continuous one. The vessels were interconnected by wing-like structures, composed of basal lamina-components: a delicate network forming nests for cells. Cells immunostained with glutamine synthetase, S100-protein or neurofilament protein contacted the vessels, as well as GFAP- or aquaporin-immunostained astrocytes. Within the body a smaller, proximal, GFAP-and aquaporin-containing subdivision, and a larger, distal, GFAP-and aquaporin-free subdivision could be distinguished. The vascular localization of agrin and utrophin, as well as dystrophin, delineated vessels unequally, preferring the proximal or distal end of the body, respectively.

  10. SparseLeap: Efficient Empty Space Skipping for Large-Scale Volume Rendering

    KAUST Repository

    Hadwiger, Markus

    2017-08-28

    Recent advances in data acquisition produce volume data of very high resolution and large size, such as terabyte-sized microscopy volumes. These data often contain many fine and intricate structures, which pose huge challenges for volume rendering, and make it particularly important to efficiently skip empty space. This paper addresses two major challenges: (1) The complexity of large volumes containing fine structures often leads to highly fragmented space subdivisions that make empty regions hard to skip efficiently. (2) The classification of space into empty and non-empty regions changes frequently, because the user or the evaluation of an interactive query activate a different set of objects, which makes it unfeasible to pre-compute a well-adapted space subdivision. We describe the novel SparseLeap method for efficient empty space skipping in very large volumes, even around fine structures. The main performance characteristic of SparseLeap is that it moves the major cost of empty space skipping out of the ray-casting stage. We achieve this via a hybrid strategy that balances the computational load between determining empty ray segments in a rasterization (object-order) stage, and sampling non-empty volume data in the ray-casting (image-order) stage. Before ray-casting, we exploit the fast hardware rasterization of GPUs to create a ray segment list for each pixel, which identifies non-empty regions along the ray. The ray-casting stage then leaps over empty space without hierarchy traversal. Ray segment lists are created by rasterizing a set of fine-grained, view-independent bounding boxes. Frame coherence is exploited by re-using the same bounding boxes unless the set of active objects changes. We show that SparseLeap scales better to large, sparse data than standard octree empty space skipping.

  11. First Exon Length Controls Active Chromatin Signatures and Transcription

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    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  12. Intron Retention and TE Exonization Events in ZRANB2

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    Sang-Je Park

    2012-01-01

    Full Text Available The Zinc finger, RAN-binding domain-containing protein 2 (ZRANB2, contains arginine/serine-rich (RS domains that mediate its function in the regulation of alternative splicing. The ZRANB2 gene contains 2 LINE elements (L3b, Plat_L3 between the 9th and 10th exons. We identified the exonization event of a LINE element (Plat_L3. Using genomic PCR, RT-PCR amplification, and sequencing of primate DNA and RNA samples, we analyzed the evolutionary features of ZRANB2 transcripts. The results indicated that 2 of the LINE elements were integrated in human and all of the tested primate samples (hominoids: 3 species; Old World monkey: 8 species; New World monkey: 6 species; prosimian: 1 species. Human, rhesus monkey, crab-eating monkey, African-green monkey, and marmoset harbor the exon derived from LINE element (Plat_L3. RT-PCR amplification revealed the long transcripts and their differential expression patterns. Intriguingly, these long transcripts were abundantly expressed in Old World monkey lineages (rhesus, crab-eating, and African-green monkeys and were expressed via intron retention (IR. Thus, the ZRANB2 gene produces 3 transcript variants in which the Cterminus varies by transposable elements (TEs exonization and IR mechanisms. Therefore, ZRANB2 is valuable for investigating the evolutionary mechanisms of TE exonization and IR during primate evolution.

  13. Recombinant Exon-Encoded Resilins for Elastomeric Biomaterials

    Science.gov (United States)

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Arinus, Shira B.; Dgany, Or; Shoseyov, Oded; Kaplan, David L.

    2011-01-01

    Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediateed dityrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative dityrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in-vivo applications of resilin biomaterials. PMID:21963157

  14. Increased constitutive nitric oxide production by whole body periodic acceleration ameliorates alterations in cardiomyocytes associated with utrophin/dystrophin deficiency.

    Science.gov (United States)

    Lopez, Jose R; Kolster, Juan; Zhang, Rui; Adams, Jose

    2017-07-01

    Duchenne Muscular Dystrophy (DMD) cardiomyopathy is a progressive lethal disease caused by the lack of the dystrophin protein in the heart. The most widely used animal model of DMD is the dystrophin-deficient mdx mouse; however, these mice exhibit a mild dystrophic phenotype with heart failure only late in life. In contrast, mice deficient for both dystrophin and utrophin (mdx/utrn(-/-), or dKO) can be used to model severe DMD cardiomyopathy where pathophysiological indicators of heart failure are detectable by 8-10weeks of age. Nitric oxide (NO) is an important signaling molecule involved in vital functions of regulating rhythm, contractility, and microcirculation of the heart, and constitutive NO production affects the function of proteins involved in excitation-contraction coupling. In this study, we explored the efficacy of enhancing NO production as a therapeutic strategy for treating DMD cardiomyopathy using the dKO mouse model of DMD. Specifically, NO production was induced via whole body periodic acceleration (pGz), a novel non-pharmacologic intervention which enhances NO synthase (NOS) activity through sinusoidal motion of the body in a headward-footward direction, introducing pulsatile shear stress to the vascular endothelium and cardiomyocyte plasma membrane. Male dKO mice were randomized at 8weeks of age to receive daily pGz (480cpm, Gz±3.0m/s(2), 1h/d) for 4weeks or no treatment, and a separate age-matched group of WT animals (pGz-treated and untreated) served as non-diseased controls. At the conclusion of the protocol, cardiomyocytes from untreated dKO animals had, respectively, 4.3-fold and 3.5-fold higher diastolic resting concentration of Ca(2+) ([Ca(2+)]d) and Na(+) ([Na(+)]d) compared to WT, while pGz treatment significantly reduced these levels. For dKO cardiomyocytes, pGz treatment also improved the depressed contractile function, decreased oxidative stress, blunted the elevation in calpain activity, and mitigated the abnormal increase in [Ca

  15. Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing.

    Science.gov (United States)

    Wongpalee, Somsakul Pop; Vashisht, Ajay; Sharma, Shalini; Chui, Darryl; Wohlschlegel, James A; Black, Douglas L

    2016-11-24

    Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest.

  16. Automatic detection of exonic splicing enhancers (ESEs using SVMs

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    Suhai Sándor

    2008-09-01

    Full Text Available Abstract Background Exonic splicing enhancers (ESEs activate nearby splice sites and promote the inclusion (vs. exclusion of exons in which they reside, while being a binding site for SR proteins. To study the impact of ESEs on alternative splicing it would be useful to have a possibility to detect them in exons. Identifying SR protein-binding sites in human DNA sequences by machine learning techniques is a formidable task, since the exon sequences are also constrained by their functional role in coding for proteins. Results The choice of training examples needed for machine learning approaches is difficult since there are only few exact locations of human ESEs described in the literature which could be considered as positive examples. Additionally, it is unclear which sequences are suitable as negative examples. Therefore, we developed a motif-oriented data-extraction method that extracts exon sequences around experimentally or theoretically determined ESE patterns. Positive examples are restricted by heuristics based on known properties of ESEs, e.g. location in the vicinity of a splice site, whereas negative examples are taken in the same way from the middle of long exons. We show that a suitably chosen SVM using optimized sequence kernels (e.g., combined oligo kernel can extract meaningful properties from these training examples. Once the classifier is trained, every potential ESE sequence can be passed to the SVM for verification. Using SVMs with the combined oligo kernel yields a high accuracy of about 90 percent and well interpretable parameters. Conclusion The motif-oriented data-extraction method seems to produce consistent training and test data leading to good classification rates and thus allows verification of potential ESE motifs. The best results were obtained using an SVM with the combined oligo kernel, while oligo kernels with oligomers of a certain length could be used to extract relevant features.

  17. Defects in mitochondrial ATP synthesis in dystrophin-deficient mdx skeletal muscles may be caused by complex I insufficiency.

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    Emma Rybalka

    Full Text Available Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca2+ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca2+ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca2+ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb's cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca2+ load in comparison to controls, and 400 nM extramitochondrial Ca2+ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.

  18. Evaluation of skeletal and cardiac muscle function after chronic administration of thymosin beta-4 in the dystrophin deficient mouse.

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    Christopher F Spurney

    Full Text Available Thymosin beta-4 (Tbeta4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tbeta4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ and mdx mice, 8-10 weeks old, were treated with 150 microg of Tbeta4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tbeta4 and amount of fibrosis were quantified using immunohistochemistry and Gomori's tri-chrome staining, respectively. Mdx mice treated with Tbeta4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tbeta4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tbeta4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.

  19. Early manifestation of alteration in cardiac function in dystrophin deficient mdx mouse using 3D CMR tagging

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    Zhong Jia

    2009-10-01

    Full Text Available Abstract Background Duchenne muscular dystrophy (DMD is caused by the absence of the cytoskeletal protein, dystrophin. In DMD patients, dilated cardiomyopathy leading to heart failure may occur during adolescence. However, early cardiac dysfunction is frequently undetected due to physical inactivity and generalized debilitation. The objective of this study is to determine the time course of cardiac functional alterations in mdx mouse, a mouse model of DMD, by evaluating regional ventricular function with CMR tagging. Methods In vivo myocardial function was evaluated by 3D CMR tagging in mdx mice at early (2 months, middle (7 months and late (10 months stages of disease development. Global cardiac function, regional myocardial wall strains, and ventricular torsion were quantified. Myocardial lesions were assessed with Masson's trichrome staining. Results Global contractile indexes were similar between mdx and C57BL/6 mice in each age group. Histology analysis showed that young mdx mice were free of myocardial lesions. Interstitial fibrosis was present in 7 month mdx mice, with further development into patches or transmural lesions at 10 months of age. As a result, 10 month mdx mice showed significantly reduced regional strain and torsion. However, young mdx mice showed an unexpected increase in regional strain and torsion, while 7 month mdx mice displayed similar regional ventricular function as the controls. Conclusion Despite normal global ventricular function, CMR tagging detected a biphasic change in myocardial wall strain and torsion, with an initial increase at young age followed by progressive decrease at older ages. These results suggest that CMR tagging can provide more sensitive measures of functional alterations than global functional indexes in dystrophin-related cardiomyopathies.

  20. Motor physical therapy affects muscle collagen type I and decreases gait speed in dystrophin-deficient dogs.

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    Thaís P Gaiad

    Full Text Available Golden Retriever Muscular Dystrophy (GRMD is a dystrophin-deficient canine model genetically homologous to Duchenne Muscular Dystrophy (DMD in humans. Muscular fibrosis secondary to cycles of degeneration/regeneration of dystrophic muscle tissue and muscular weakness leads to biomechanical adaptation that impairs the quality of gait. Physical therapy (PT is one of the supportive therapies available for DMD, however, motor PT approaches have controversial recommendations and there is no consensus regarding the type and intensity of physical therapy. In this study we investigated the effect of physical therapy on gait biomechanics and muscular collagen deposition types I and III in dystrophin-deficient dogs. Two dystrophic dogs (treated dogs-TD underwent a PT protocol of active walking exercise, 3×/week, 40 minutes/day, 12 weeks. Two dystrophic control dogs (CD maintained their routine of activities of daily living. At t0 (pre and t1 (post-physical therapy, collagen type I and III were assessed by immunohistochemistry and gait biomechanics were analyzed. Angular displacement of shoulder, elbow, carpal, hip, stifle and tarsal joint and vertical (Fy, mediolateral (Fz and craniocaudal (Fx ground reaction forces (GRF were assessed. Wilcoxon test was used to verify the difference of biomechanical variables between t0 and t1, considering p<.05. Type I collagen of endomysium suffered the influence of PT, as well as gait speed that had decreased from t0 to t1 (p<.000. The PT protocol employed accelerates morphological alterations on dystrophic muscle and promotes a slower velocity of gait. Control dogs which maintained their routine of activities of daily living seem to have found a better balance between movement and preservation of motor function.