In patients with Duchenne muscular dystrophy (DMD), the absence of a functional dystrophin protein results in sarcolemmal instability, abnormal calcium signaling, cardiomyopathy, and skeletal muscle ...degeneration. Using the dystrophin-deficient sapje zebrafish model, we have identified microRNAs (miRNAs) that, in comparison to our previous findings in human DMD muscle biopsies, are uniquely dysregulated in dystrophic muscle across vertebrate species. MiR-199a-5p is dysregulated in dystrophin-deficient zebrafish, mdx(5cv) mice, and human muscle biopsies. MiR-199a-5p mature miRNA sequences are transcribed from stem loop precursor miRNAs that are found within the introns of the dynamin-2 and dynamin-3 loci. The miR-199a-2 stem loop precursor transcript that gives rise to the miR-199a-5p mature transcript was found to be elevated in human dystrophic muscle. The levels of expression of miR-199a-5p are regulated in a serum response factor (SRF)-dependent manner along with myocardin-related transcription factors. Inhibition of SRF-signaling reduces miR-199a-5p transcript levels during myogenic differentiation. Manipulation of miR-199a-5p expression in human primary myoblasts and myotubes resulted in dramatic changes in cellular size, proliferation, and differentiation. MiR-199a-5p targets several myogenic cell proliferation and differentiation regulatory factors within the WNT signaling pathway, including FZD4, JAG1, and WNT2. Overexpression of miR-199a-5p in the muscles of transgenic zebrafish resulted in abnormal myofiber disruption and sarcolemmal membrane detachment, pericardial edema, and lethality. Together, these studies identify miR-199a-5p as a potential regulator of myogenesis through suppression of WNT-signaling factors that act to balance myogenic cell proliferation and differentiation.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary ...mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wildtype and production strains identified a Val-59 leads to Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311 leads to Ile mutation in the aspartokinase gene (lysC), and a Pro-458 leads to Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l-1 h-1) so far reported for microbes producing L-lysine in fed-batch fermentation.
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CEKLJ, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA) (OMIM #617481) is an autosomal recessive disease characterized by progressive microcephaly, ...plagiocephaly, hypotonia, spastic quadriparesis, global developmental delay, intellectual disability, optic features and abnormal brain magnetic resonance imaging (MRI). NMIHBA was recently reported to be caused by PRUNE1 mutations. Eight mutations have been reported in 13 unrelated families. Here, we report 3 PRUNE1 mutations in 1 Caucasian and 3 Japanese families. One recurrent missense mutation (p.Asp106Asn) was previously reported in Turkish and Italian families, while the other 2 mutations (p.Leu18Serfs*8 and p.Cys180*) are novel. We also show that mutant PRUNE1 mRNA can be subject to nonsense‐mediated mRNA decay. The patients presented in this study showed atypical NMIHBA phenotypes with no progressive microcephaly. Furthermore, one Caucasian case had significant macrocephaly; therefore, patients with PRUNE1 mutations can exhibit a broad and heterogeneous spectrum of phenotypes.
Neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA) (OMIM #617481) is an autosomal recessive disease characterized by progressive microcephaly, plagiocephaly, hypotonia, spastic quadriparesis, global developmental delay, intellectual disability, optic features and abnormal brain magnetic resonance imaging (MRI). NMIHBA was recently reported to be caused by PRUNE1 mutations. Here, we report 3 PRUNE1 mutations in 1 Caucasian and 3 Japanese families. The patients presented in this study showed atypical NMIHBA phenotypes with no progressive microcephaly. Furthermore, one Caucasian case had significant macrocephaly; therefore, patients with PRUNE1 mutations can exhibit a broad and heterogeneous spectrum of phenotypes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
MEGF10 is a single transmembrane protein that is expressed in satellite cells of skeletal muscle, with 17 EGF-like domains in the extracellular region and 13 tyrosine residues in the cytoplasmic ...domain. Recessive mutations in MEGF10 are known to cause a congenital myopathy in humans, including the syndrome of early onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD). Previously we reported a family with a milder phenotype who harbored the compound heterozygous missense mutations C326R and C774R. A heterozygous C774R mutation was also found in a patient with EMARDD, paired with a heterozygous nonsense mutation in MEGF10, suggesting that C774R may be more damaging than C326R. Both mutations reside in the extracellular EGF-like domains of MEGF10. We found that these mutations were associated with decreased tyrosine phosphorylation of MEGF10 in vitro, and that C774R caused a greater impairment of tyrosine phosphorylation than C326R. We also found that Y1030 is the major tyrosine phosphorylation site in MEGF10 and is phosphorylated at least in part by c-Src. Co-expression of dominant-negative c-Src with MEGF10 shows decreased tyrosine phosphorylation of MEGF10, suggesting that MEGF10 is phosphorylated by endogenous c-Src. Overexpression of wild-type MEGF10 enhanced C2C12 myoblast proliferation, while overexpression of Y1030F mutated MEGF10 did not. In addition, overexpression of either wild type MEGF10 or Y1030D caused mild activation of ERK1/2. These findings indicate that MEGF10-mediated signaling via tyrosine phosphorylation plays an important role in myoblast proliferation, possibly via activation of ERK1/2. We conclude that MEGF10-mediated signaling via tyrosine phosphorylation helps to regulate myoblast proliferation. Our findings suggest that defects in this signaling pathway may contribute to the disease mechanism of MEGF10 myopathy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot ...disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan. References: (1) K. Kageyama et al. J. Phytopathol. 151:485, 2003. (2) S. W. Ritchie et al. Spec. Rep. 48. Iowa State Univ. Coop Ext. Serv., Ames, 1993. (3) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.
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