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In this research, we aimed to predict the relative risk of the recent new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the basis of our previous research. We first performed molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to determine the binding affinities to human cells of three new variants of SARS-CoV-2: Omicron BQ.1, XBB, and XBB.1.5 We then investigated the three variants to discover the evolutionary distance of the spike protein gene (S gene) from the Wuhan, Omicron BA.1, and Omicron BA.4/5 variants, to understand the changes in the S gene.The results indicated that the XBB.1.5 variant had the highest binding affinity of the spike protein with ACE2 and the longest evolutionary distance of the S gene. Thisin silicoevidence suggested that the XBB.1.5 variant may produce infections that spread more widely and faster than can infections of preexisting variants.

Core body temperature (CBT) is considered a valuable marker for circadian rhythm. This study aimed to investigate the changes in CBT that are associated with the symptoms of circadian rhythm sleep-wake disorder (CRSWD) post-treatment in children.Twenty-eight school-age children [10 boys and 18 girls; mean age (±standard deviation), 13.68 ± 0.93 years] who were admitted to our hospital with CRSWD underwent treatment for 6-8 weeks according to the following protocol: lights-out for sleep at 21:00; phototherapy for waking at 6:00 or 7:00; light exercise everyday (eg, a 20- to 30-min walk). CBT was continuously measured for 24 h on the first day of admission and on the first day after treatment.The mean time of sleep onset/offset (±standard deviation; in hours:minutes) 1 week before admission and 1 week after treatment were 23:53 ± 2:26/9:58 ± 2:15 and 21:17 ± 0:19/6:46 ± 0:32, respectively. The mean times of sleep onset and offset measured post-treatment were significantly earlier than those measured pre-treatment (p 0.001). The mean CBT and mean minimum CBT during sleep were significantly lower on the first day post-treatment than on the first day of admission (p = 0.011 and p 0.001, respectively).Symptom improvements in patients with CRSWD were associated with a decrease in CBT during sleep, suggesting that CBT may be a biomarker for improvements in CRSWD. These results help elucidate the cause of this sleep disorder.

ObjectivesIn our previous research, we developed a mathematical model via molecular simulation analysis to predict the infectivity of seven SARS-CoV-2 variants. In this report, we aimed to predict the relative risk of the recent new variants of SARS-CoV-2 as based on our previous research.MethodsWe subjected Omicron BA.4/5 and BA.2.75 variants of SARS-CoV-2 to the analysis to determine the absolute evolutionary distance of the spike protein gene (S gene) of the variants from the Wuhan variant so as to appreciate the changes in the spike protein. We performed the molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to understand the docking affinities of these variants. We then compared the evolutionary distances and the docking affinities of these variants with those of the seven variants that we had analyzed in our previous research.ResultsThe evolutionary distances of the S gene in BA.4/5 and BA.2.75 from the Wuhan variant were longer than those of the other variants. BA.2.75 had the highest docking affinity of the spike protein with ACE2 (ratio per Wuhan variant).ConclusionThe important results from this analysis are the following: BA.2.75 has both the highest docking affinity and the longest evolutionary distance of the S gene. These results suggest that BA.2.75 infection can spread farther than can infections of preexisting variants.

STUDY OBJECTIVES: We aimed to investigate whether improvements in the symptoms of circadian rhythm sleep-wake disorder after treatment were associated with an increase in serum insulin-like growth factor-1 (IGF-1) concentration. METHODS: Eighty-seven school-aged children (32 males, 55 females), aged 14.31 ± 1.50 years (mean ± standard deviation), who were admitted to our hospital with circadian rhythm sleep-wake disorder received treatment for 6–8 weeks consisting of the following protocol: (1) lights-out for sleep occurred at 21:00, (2) phototherapy for waking started at 06:00 or 07:00, and (3) light exercise was required every day (eg, a 20- to 30-minute walk). Blood samples were collected at 08:00 am to measure the serum concentrations of IGF-1, pre- and posttreatment. RESULTS: The mean times of day of sleep onset and offset at the pre- and posttreatment timepoints were 23:32 ± 4.21 and 10:27 ± 2.98, and 21:26 ± 0.55 and 06:50 ± 0.70, respectively. The mean times of day of sleep onset and offset measured at the posttreatment timepoint were significantly earlier compared with the pretreatment baselines (P < .01). The mean serum levels of IGF-1 significantly increased from 315.59 ± 68.26 ng/mL at pretreatment to 335.09 ± 69.78 ng/mL at posttreatment (P < .01). CONCLUSIONS: Improvements in the symptoms of patients with circadian rhythm sleep-wake disorders were associated with increased serum concentrations of IGF-1, suggesting that serum IGF-1 may be a biomarker of improvements in school-aged children with circadian rhythm sleep-wake disorder. CITATION: Kimura S, Toyoura M, Toyota Y, Takaoka Y. Serum concentrations of insulin-like growth factor-1 as a biomarker of improved circadian rhythm sleep-wake disorder in school-aged children. J Clin Sleep Med. 2020;16(12):2073–2078.

Variants of a coronavirus (SARS-CoV-2) have been spreading in a global pandemic. Improved understanding of the infectivity of future new variants is important so that effective countermeasures against them can be quickly undertaken. In our research reported here, we aimed to predict the infectivity of SARS-CoV-2 by using a mathematical model with molecular simulation analysis, and we used phylogenetic analysis to determine the evolutionary distance of the spike protein gene (S gene) of SARS-CoV-2. We subjected the six variants and the wild type of spike protein and human angiotensin-converting enzyme 2 (ACE2) to molecular docking simulation analyses to understand the binding affinity of spike protein and ACE2. We then utilized regression analysis of the correlation coefficient of the mathematical model and the infectivity of SARS-CoV-2 to predict infectivity. The evolutionary distance of the S gene correlated with the infectivity of SARS-CoV-2 variants. The coefficient of the mathematical model obtained with results of molecular docking simulation also correlated with the infectivity of SARS-CoV-2 variants. These results suggest that the data from the docking simulation for the receptor binding domain of variant spike proteins and human ACE2 were valuable for prediction of SARS-CoV-2 infectivity. In addition, we developed a mathematical model for prediction of SARS-CoV-2 variant infectivity by using binding affinity obtained via molecular docking and the evolutionary distance of the S gene.

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the 'molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24 + 6) families (83.3%). We identified eight mutated genes in 27 (21 + 6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T>A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.

Pompe disease is caused by an inborn defect of lysosomal acid α-glucosidase (GAA) and is characterized by lysosomal glycogen accumulation primarily in the skeletal muscle and heart. Patients with the severe type of the disease, infantile-onset Pompe disease (IOPD), show generalized muscle weakness and heart failure in early infancy. They cannot survive over two years. Enzyme replacement therapy with recombinant human GAA (rhGAA) improves the survival rate, but its effect on skeletal muscle is insufficient compared to other organs. Moreover, the patho-mechanism of skeletal muscle damage in IOPD is still unclear. Here we generated induced pluripotent stem cells (iPSCs) from patients with IOPD and differentiated them into myocytes. Differentiated myocytes showed lysosomal glycogen accumulation, which was dose-dependently rescued by rhGAA. We further demonstrated that mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activity was impaired in IOPD iPSC-derived myocytes. Comprehensive metabolomic and transcriptomic analyses suggested the disturbance of mTORC1-related signaling, including deteriorated energy status and suppressed mitochondrial oxidative function. In summary, we successfully established an in vitro skeletal muscle model of IOPD using patient-specific iPSCs. Disturbed mTORC1 signaling may contribute to the pathogenesis of skeletal muscle damage in IOPD, and may be a potential therapeutic target for Pompe disease.

Background The purpose of this study was to evaluate the utility of a wrist actigraph for estimating muscle strength in Duchenne muscular dystrophy patients. Methods Twenty-two patients aged 4–19 years wore a wrist actigraph to monitor activities of daily living, and underwent a test of knee extension strength and the 6 min walk test. These measures were made at baseline and at 1 year later. The actigraph data were quantified using the zero crossing mode (ZCM), which indicates the frequency of movement, and the proportional integration mode (PIM), which indicates activity level or vigor of motion. Results The ZCM and PIM scores of ambulatory patients were higher than those of non-ambulatory patients (P < 0.001). The correlation coefficient between ZCM score and 6 min walk distance, ZCM score and knee extension strength, PIM score and 6 minute walk distance, and PIM score and knee extension strength was –0.44, 0.25, 0.58, and 0.63, respectively. This indicates that the PIM score had a moderate–good association with 6 min walk distance and knee extension strength. Conclusion Muscle strength can be estimated using the PIM score calculated from actigraph data. The PIM score is a good tool for the estimation of muscle strength.

Hashimoto's encephalopathy is an anti-thyroid antibody-positive autoimmune encephalopathy. We herein report the case of a 13-year-old male patient with subacute vertigo, muscle weakness in the extremities and gait disturbance who was diagnosed with Hashimoto's encephalopathy. He showed no severe impairment of consciousness and no seizures, and there were no abnormalities on the brain MRI. However, epileptic spike and wave complexes were observed on an electroencephalogram, and a decline in blood flow was diffusely observed on brain SPECT (single photon emission computed tomography). His thyroid function was normal, but he was positive for anti-thyroid antibodies, such as anti-TPO (thyroid peroxidase) antibodies. He was also positive for serum anti-NAE (NH2-terminal alpha-enolase) antibodies. Systemic corticosteroid therapy and high-dose intravenous immunoglobulin therapy were effective, greatly improving his quality of life.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial. Methods: Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription–polymerase chain reaction. Results: Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event. Conclusion: An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.

Duchenne muscular dystrophy (DMD) is caused by mutation of the dystrophin gene. Cases of dystrophinopathy with a 2-bp deletion in the dystrophin gene commonly result in DMD. We report here a case of dystrophinopathy in a 9-years-old boy with a 2-bp deletion in exon 74 of the dystrophin gene; however, the boy had no clear clinical signs of muscle weakness. Immunohistochemical studies with N-terminal (DYS3) and rod-domain anti-dystrophin (DYS1) antibodies revealed that the dystrophin signals were weaker than in the control sample (non-dystrophinopathy) at the sarcolemma of myofibers, and the studies with C-terminus anti-dystrophin antibody (DYS2) were negative. Our patient’s mutation is located between the binding sites of α-syntrophin and α-dystrobrevin. These results suggest that this mutation does not clearly induce muscle weakness at least through the age of 9 years.

Duchenne/Becker muscular dystrophy is a progressive muscle disease, which is caused by the abnormality of dystrophin. Spina bifida is characterized by paralysis of the feet, with most of the upper extremities not being affected. We report here on the first case of Becker muscular dystrophy coinciding with spina bifida. The muscle biopsy specimens of the patient showed dystrophic changes in upper extremities, but clearly less in lower extremities. The results show that the restriction of excessive exercise is important for dystrophin deficiency disease.

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Therapeutic options for DMD are limited because the pathogenetic mechanism by which dystrophin deficiency produces the clinical phenotype remains obscure. Recent reports of abnormal alpha-adrenergic vasoregulation in the exercising muscles of DMD patients and in the mdx mouse, an animal model of DMD, prompted us to hypothesize that the dystrophin-deficient smooth muscle contributes to the vascular and dystrophic phenotypes of DMD. To test this, we generated transgenic mdx mice that express dystrophin only in smooth muscle (SMTg/mdx). We found that alpha-adrenergic vasoconstriction was markedly attenuated in the contracting hindlimbs of C57BL/10 wild-type mice, an effect that was mediated by nitric oxide (NO) and was severely impaired in the mdx mice. SMTg/mdx mice showed an intermediate phenotype, with partial restoration of the NO-dependent modulation of alpha-adrenergic vasoconstriction in active muscle. In addition, the elevated serum creatine kinase levels observed in mdx mice were significantly reduced in SMTg/mdx mice. This is the first report of a functional role of dystrophin in vascular smooth muscle.

Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.

Aminoglycoside antibiotics have been found to suppress nonsense mutations located in the defective dystophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD). However, it is very difficult to find patients that are applicable for this therapy, because: (1) only 5-13% of DMD patients have nonsense mutations in the dystrophin gene, (2) it is challenging to find nonsense mutations in the gene because dystrophin cDNA is very long (14 kb), and (3) the efficiency of aminoglycoside-induced read-through is dependent on the kind of nonsense mutation. In order to develop a system for identifying candidates that qualify for aminoglycoside therapy, fibroblasts from nine DMD patients with nonsense mutation of dystrophin gene were isolated, induced to differentiate to myogenic lineage by AdMyoD, and exposed with gentamicin. The dystrophin expression in gentamicin-exposed myotubes was monitored by in vitro dystrophin staining and western blotting analysis. The results showed that gentamicin was able to induce dystrophin expression in the differentiated myotubes by the read-through of the nonsense mutation TGA in the gene; a read-through of the nonsense mutations TAA and TAG did not occur and consequently did not lead to dystrophin expression. Therefore, it is speculated that the aminoglycoside treatment is far more effective for DMD patients that have nonsense mutation TGA than for patients that have nonsense mutation TAA and TAG. In this study, we introduce an easy system to identify patients for this therapy and report for the first time, that dystrophin expression was detected in myotubes of DMD patients using gentamicin.

Duchenne's muscular dystrophy (DMD) is a lethal muscle disease caused by a lack of dystrophin expression at the sarcolemma of muscle fibers. We investigated retroviral vector delivery of dystrophin in dystrophin-deficient DMD(mdx) (hereafter referred to as mdx) mice via an ex vivo approach using mdx muscle-derived stem cells (MDSCs). We generated a retrovirus carrying a functional human mini-dystrophin (RetroDys3999) and used it to stably transduce mdx MDSCs obtained by the preplate technique (MD3999). These MD3999 cells expressed dystrophin and continued to express stem cell markers, including CD34 and Sca-1. MD3999 cells injected into mdx mouse skeletal muscle were able to deliver dystrophin. Though a relatively low number of dystrophin-positive myofibers was generated within the gastrocnemius muscle, these fibers persisted for up to 24 weeks postinjection. The injection of cells from additional MDSC/Dys3999 clones into mdx skeletal muscle resulted in varying numbers of dystrophin-positive myofibers, suggesting a differential regenerating capacity among the clones. At 2 and 4 weeks postinjection, the infiltration of CD4- and CD8-positive lymphocytes and a variety of cytokines was detected within the injected site. These data suggest that the transplantation of retrovirally transduced mdx MDSCs can enable persistent dystrophin restoration in mdx skeletal muscle; however, the differential regenerating capacity observed among the MDSC/Dys3999 clones and the postinjection immune response are potential challenges facing this technology.

The ZHTc6-MyoD embryonic stem cell line expresses the myogenic transcriptional factor MyoD under the control of a tetracycline-inducible promoter. Following induction, most of the ZHTc6-MyoD cells differentiate to myotubes. However, a small fraction does not differentiate, instead forming colonies that retain the potential for myocyte differentiation. In our current study, we found that parathyroid hormone type 1 receptor (PTH1R) expression in colony-forming cells at 13 days after differentiation was higher than that in the undifferentiated ZHTc6-MyoD cells. We also found that PTH1R expression was required for myocyte differentiation, and that parathyroid hormone accelerated the differentiation. Our analysis of human and mouse skeletal muscle tissues showed that most cells expressing PTH1R also expressed Pax7 and CD34, which are biomarkers of satellite cells. Furthermore, we found that parathyroid hormone treatment significantly improved muscle weakness in dystrophin-deficient mdx mice. This is the first report indicating that PTH1R and PTH accelerate myocyte differentiation.

The purpose of this study was to evaluate the utility of a wrist actigraph for estimating muscle strength in Duchenne muscular dystrophy patients.Twenty-two patients aged 4-19 years wore a wrist actigraph to monitor activities of daily living, and underwent a test of knee extension strength and the 6 min walk test. These measures were made at baseline and at 1 year later. The actigraph data were quantified using the zero crossing mode (ZCM), which indicates the frequency of movement, and the proportional integration mode (PIM), which indicates activity level or vigor of motion.The ZCM and PIM scores of ambulatory patients were higher than those of non-ambulatory patients (P0.001). The correlation coefficient between ZCM score and 6 min walk distance, ZCM score and knee extension strength, PIM score and 6 minute walk distance, and PIM score and knee extension strength was -0.44, 0.25, 0.58, and 0.63, respectively. This indicates that the PIM score had a moderate-good association with 6 min walk distance and knee extension strength.Muscle strength can be estimated using the PIM score calculated from actigraph data. The PIM score is a good tool for the estimation of muscle strength.

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin+ cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34+ cells and Bcl-2+ cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34+Bcl-2+ enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.

Myoclonic epilepsy associated with ragged-red fibers is one of the mitochondrial encephalomyopathies. Pathogenic mitochondrial DNA mutations have been identified in the mitochondrial transfer RNA (tRNA)(Lys) at positions 8344 and 8356. Characteristics of myoclonic epilepsy associated with ragged-red fibers include myoclonic epilepsy, generalized epilepsy, hearing loss, exercise intolerance, lactic acidosis, and ragged-red fibers. The elevated lactate level is one of the most important symptoms needed to make a diagnosis of mitochondrial encephalomyopathy. In the present case, however, myoclonic epilepsy was associated with ragged-red fibers but without increased lactate levels. Therefore, myoclonic epilepsy associated with ragged-red fibers should be suspected in a patient who has myoclonic epilepsy that is difficult to control with antiepileptic medications and who has other symptoms of mitochondrial disease, such as mental retardation, even if the patient's lactate level is normal.

We examined the levels of soluble CD23 (sCD23) and IL-5 in the serum and culture supernatants of the peripheral blood mononuclear cells of a 9-year-old girl with cutaneous paragonimiasis without respiratory symptoms. Before treatment, levels of both sCD23 and IL-5 in her serum and culture supernatants were elevated compared with those of controls. After successful treatment with praziquantel, sCD23 and IL-5 levels rapidly reduced to normal levels. These results indicate that sCD23 and IL-5 are involved in the immune-mediated pathological responses seen in paragonimiasis.

In order to study the regulatory mechanism of developmental and tissue-specific expression of the muscle type dystrophin gene in mice, transgenic mice were generated carrying the 900 bp genomic fragment derived from the muscle type dystrophin promoter region fused to the bacterial lacZ gene. Six independent transgenic mouse lines showed specific reporter gene expression in the right heart, but not in skeletal or smooth muscle. The reporter gene expression was first detected in the presumptive right ventricle of the embryos at 8.5 days post coitum and the expression continued only in the right ventricle throughout the development and at the adult stage. The results indicate that the 900 bp genomic fragment contains the regulatory element required for expression of dystrophin only in the right heart, suggesting that distinct elements are responsible for the expression in the left and right compartments of the heart, and/or in skeletal and smooth muscle cells. Based on these findings, the relationship between defects in muscle type promoter and the diseases caused by abnormal dystrophin expression is discussed.

The Escherichia coli lacZ gene has frequently been used as a reporter in cell lineage analysis, in determining the elements regulating spatial and temporal gene expression, and in enhancer/gene trap detection of developmentally regulated genes. However, it is uncertain whether lacZ expression affects eukaryotic cell growth and development. By using a gene trap, we previously isolated the promoter, Ayu1, which is active in ES cells and in several tissues including the gonads. We used this promoter and the nuclear location signal of the SV40 large T gene to locate β-galactosidase either in the cytoplasm or the nucleus. Transgenic lines containing β-galactosidase in the cytoplasm of a wide variety of cell types did not transmit the transgene to their offspring. In contrast, transgenic mice, containing β-galactosidase in the nucleus, did transmit the transgene successfully. Interestingly, lacZ expression in the brain was more restricted when β-galactosidase activity was detected in the cytoplasm. These data suggested that cytoplasmic β-galactosidase affects certain developmental processes or gametogenesis resulting in transmission distortion of the transgene, and that this effect can be reduced by targeting β-galactosidase to the nucleus. We also found that Ayu1-driven lacZ expression in the duodenum of adult transgenic mice was sexually dimorphic, being positive in females and negative in males.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial.Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription-polymerase chain reaction.Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event.An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.

New trap vectors (U1 and U2) have been developed to trap genes in murine embryonic stem (ES) cells. The polyA addition signal of the neomycin phosphotransferase II (neo) gene was removed from these vectors so that they needed to trap an endogenous polyA signal for expression of the neo gene. The frequency of gene-trap events of these vectors was about five times higher than with the vector containing the polyA signal, and only one copy of the trap vector was integrated in most cases. Four out of five 5'-flanking regions of the integrated vector in ES cell lines were found to be novel endogenous promoters, suggesting that this method is efficient for trapping genes in ES cells. In two cases analyzed, large deletions or rearrangements spanning more than 10 kb were found in the 3'-flanking region of the trap vector introduced by electroporation. This result suggests that phenotypes observed in homozygotes with a mutated allele could be due to the disruption of a gene adjacent to the trapped gene, but not of the trapped gene.

Purpose: A clinical trial was conducted to evaluate the safety and efficacy of alglucosidase alfa in infants and children with advanced Pompe disease. Methods: Open-label, multicenter study of IV alglucosidase alfa treatment in 21 infants 3–43 months old (median 13 months) with minimal acid α-glucosidase activity and abnormal left ventricular mass index by echocardiography. Patients received IV alglucosidase alfa every 2 weeks for up to 168 weeks (median 120 weeks). Survival results were compared with an untreated reference cohort. Results: At study end, 71% (15/21) of patients were alive and 44% (7/16) of invasive-ventilator free patients remained so. Compared with the untreated reference cohort, alglucosidase alfa reduced the risk of death by 79% (P < 0.001) and the risk of invasive ventilation by 58% (P = 0.02). Left ventricular mass index improved or remained normal in all patients evaluated beyond 12 weeks; 62% (13/21) achieved new motor milestones. Five patients were walking independently at the end of the study and 86% (18/21) gained functional independence skills. Overall, 52% (11/21) of patients experienced infusion-associated reactions; 95% (19/20) developed IgG antibodies to recombinant human lysosomal acid α-glucosidase; no patients withdrew from the study because of safety concerns. Conclusions: In this population of infants with advanced disease, biweekly infusions with alglucosidase alfa prolonged survival and invasive ventilation-free survival. Treatment also improved indices of cardiomyopathy, motor skills, and functional independence.

One cause of X-linked dilated cardiomyopathies is mutation of the dystrophin gene. We report the case of a young boy who suffered from dilated cardiomyopathy caused only by dystrophin-deficient cardiac muscle, but who did not present with any clinical signs of skeletal myopathy. Sequence analysis of the patient's dystrophin gene revealed the presence of a novel single point mutation at the first exon—intron boundary, inactivating the 5′ splice site consensus sequence of the first intron. The lack of muscle weakness observed clinically can be explained by expression of the brain and Purkinje dystrophin isoforms in skeletal muscle.

We established genetically engineered ES (ZHTc6-MyoD) cells that harbor a tetracycline-regulated expression vector encoding myogenic transcriptional factor MyoD, for the therapy of muscle diseases, especially Duchenne muscular dystrophy (DMD). Almost all the ZHTc6-MyoD cells were induced into muscle lineage after removal of tetracycline. The undifferentiated ZHTc6-MyoD cells are Sca-1+ and c-kit+, but CD34-, all well-known markers for mouse hematopoietic stem cells. In addition, they are able to maintain themselves in the undifferentiated state, even after one month of culture. Therefore, it is possible to obtain a large quantity of ZHTc6-MyoD cells in the undifferentiated state that maintain the potential to differentiate only into muscle lineage. Additionally, at two weeks post-injection of these cells into muscle of mdx, a model mouse of DMD, clusters of dystrophin-positive myofibers were observed at the injection site. Therefore, ES cells have considerable therapeutic potential for treating muscle diseases.

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC) and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

Epsilon-polylysine is a homopolymer of L-lysine, containing approximately 30 L-lysine subunits, as synthesized in aerobic bacterial fermentation by Streptomyces albulus. epsilon -Polylysine is approved for food use in Japan as an antimicrobial preservative. A series of pharmacokinetic and metabolic profile studies on epsilon -polylysine have been conducted in rats in order to provide a better understanding of the reason for its lack of toxicological effects in subchronic and chronic feeding bioassays using relatively high concentrations in the diet up to 50000 ppm. As reported in this article, epsilon -polylysine was practically non-toxic in an acute oral toxicity study in rats, with no mortality up to 5 g/kg and was not mutagenic in bacterial reversion assays. Absorption, distribution, metabolism and excretion (ADME) studies on 14C-radiolabeled epsilon -polylysine, given in a single dose to fasted male rats at 100mg/kg, revealed low absorption from the gastrointestinal tract. All but trace amounts of the dosed radioactivity was eliminated by excretion within 168 h and over 97% was accounted for in urine (1.2%), feces (92.9%), or expired air (3%) by 48 h. The sum of the cumulative excretion with routes associated with absorption in urine, expired air and carcass was 6.4% of total recovered radioactivity; approximately 94% of the dose of epsilon -polylysine passed unabsorbed through the gastrointestinal tract in the feces. Whole body autoradiography did not show concentration of absorbed epsilon -polylysine in any tissue or organ. Excretion half-lives of epsilon -polylysine equivalents in blood and plasma were 20 and 3.9 days, likely prolonged by the incorporation into protein of cleaved L-lysine. Metabolic profiles by HPLC analysis of plasma samples suggest that L-lysine is the predominant early metabolic by-product, likely from protease activity in the upper GI tract; only 0.2% of the administered parent compound was found in plasma. At 8-72 h, HPLC profiles show diminishing levels of epsilon -polylysine and L-lysine in plasma, accompanied by a shift to larger peaks of homopolymer fragments of varying subunit length, presumably from microbial degradation of epsilon -polylysine in the lower gut. HPLC profiles of urine and feces collected from 0 to 24 h post-dosing revealed three distinct peaks in urine, the first peak likely to be epsilon -polylysine and epsilon -polylysine less a few amino acid subunits, and the second, L-lysine and the third, a metabolite of L-lysine. Radiolabeled L-lysine was reduced from 67.2% of the radioactivity in plasma at 30 min to 7.5% at 4 h, indicating that L-lysine is readily removed from plasma from essential amino acid incorporation into protein. Based on the findings of the ADME studies and lack of toxicity in safety studies, the proposed use of epsilon -polylysine as a preservative in foods is considered to be safe.

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.

In previous transgenic studies, we reported a 0.9 kb fragment from a mouse dystrophin muscle promoter that contains the regulatory elements required for expression of dystrophin only in the right heart. In this study, to further characterize the regulation of muscle type of promoter, we analyzed promoter activity and tissue specificity using a total 14 kb fragment around the human dystrophin muscular-specific exon 1 in vitro and in vivo. In vitro analysis showed that the lacZ construct of the 7 kb promoter and 7 kb intron 1 was expressed 2.5 times as strong as the lacZ construct of only the 7 kb promoter in C2/4 myotubes. In vivo analysis revealed expression of both constructs in the whole heart, skeletal muscle and vascular smooth muscle in embryos. However, in adults, the expression in skeletal muscle disappeared. We conclude that the 7 kb upstream region and the 7 kb intronic region included responsible elements for the expression in the heart, but not in skeletal muscle in vivo. It is possible that a strong enhancer element for skeletal muscle exists in some other region.

We report on the first case of X-linked recessive myotubular myopathy (MTM1) coinciding with Duchenne muscular dystrophy (DMD). The muscle biopsy specimens of the patient show only the characteristic findings of MTM1, without the findings of DMD. We theorize that this was caused by the muscle fiber immaturity and inactivity.

Duchenne muscular dystrophy is a muscle-wasting disease accompanied by a variable, but often significant degree of mental retardation, possibly due to the absence of dystrophin. However, the function of brain type dystrophin remains insufficiently clear. With this background, in order to study the cell-specific regulation of brain type dystrophin expression in mice, we generated transgenic mice carrying the 2.1 kb 5'-fragment of the mouse brain type dystrophin gene, fused to the coding region of the bacterial lacZ gene. Three transgenic mice lines showed lacZ expression in the cerebral cortex. However, lacZ expression was not detected in the CA region of the hippocampus. These results suggest that the 2.1 kb 5'-fragment of the mouse brain type dystrophin gene contains the regulatory element required for its expression in the cerebral cortex, but not in the hippocampus.

Introduction: Duchenne muscular dystrophy (DMD) is a severe muscle degenerative disorder caused by mutations in the dystrophin gene. Embryonic stem cell (ES cell) transplantation is one of the more promising therapies because ES cells are available in large quantities and can serve to systemically restore affected muscles of DMD patients. Although ES cells have shown to be capable of differentiating into various tissues and cell types, it has been difficult to induce differentiation of ES cells specifically into muscle fibers only. We are proposing a method to overcome this impediment by establishing genetically engineered ES cells which harbor a tetracycline-regulated expression vector encoding the myogenic transcriptional factor MyoD.

Duchenne muscular dystrophy (DMD) is an X-linked fatal disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin, which is associated with a complex of proteins that spans the membrane and effectively links the cytoskeleton to the extracellular matrix. Despite the abundance of information about the molecular basis of this disease, there is currently no effective treatment for DMD because the mechanism by which dystrophin deficiency produces the clinical phenotype is poorly understood. Our previous data showed that dystrophin expression is normally detectable in smooth muscle cells (SMCs) as well as in skeletal muscle cells. Likewise, it is defective in SMCs of DMD patients and mdx mice, an animal model of DMD. Recent studies have revealed that focal multicellular myocytolytic lesions in muscular dystrophies may represent functional vascular abnormalities, regardless of whether or not they are primary. Although vascular dysfunction in DMD patients and mdx mice has been also reported, it is considered that the dystrophin deficiency of skeletal muscle and/or vascular endothelium, not vascular smooth muscle cells (VSMCs), is responsible for the vascular abnormalities.

Aminoglycoside antibiotics have been found to suppress nonsense mutations located in the defective dystrophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD). However, the gentamicin therapy is hindered by several problems. First, a high dose of gentamicin, which is needed to induce a read-through of the nonsense mutations in DMD patients, can not be given because of oto- and nephrotoxicity. Even if this dose could be tolerated, this high concentration cannot be sustained due to the short half-life of gentamicin. Lastly, identifying patients that are applicable for this therapy is difficult because: 1) only 5 to 13% of DMD patients have nonsense mutations in the dystrophin gene, 2) it is challenging to find nonsense mutations in the gene because dystrophin cDNA is very long (14kb), and 3) the efficiency of aminoglycoside-induced read-through is dependent on the kind of nonsense mutation.