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Chitin deacetylase (CDA) is a chitin degradation enzyme that catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues, playing an important role in the high-value utilization of waste chitin. The shells of shrimp and crab are rich in chitin, and mangroves are usually recognized as an active habitat to shrimp and crab. In the present study, a CDA-producing bacterium, strain TCI-16, was isolated and screened from the mangrove soil. Strain TCI-16 was identified and named as Bacillus aryabhattai TCI-16, and the maximum CDA activity in fermentation broth reached 120.35 ± 2.40 U/mL at 36 h of cultivation. Furthermore, the complete genome analysis of B. aryabhattai TCI-16 revealed the chitin-degrading enzyme system at genetic level, in which a total of 13 putative genes were associated with carbohydrate esterase 4 (CE4) family enzymes, including one gene coding CDA, seven genes encoding polysaccharide deacetylases, and five genes encoding peptidoglycan-N-acetyl glucosamine deacetylases. Amino acid sequence analysis showed that the predicted CDA of B. aryabhattai TCI-16 was composed of 236 amino acid residues with a molecular weight of 27.3 kDa, which possessed a conserved CDA active like the known CDAs. However, the CDA of B. aryabhattai TCI-16 showed low homology (approximately 30%) with other microbial CDAs, and its phylogenetic tree belonged to a separate clade in bacteria, suggesting a high probability in structural novelty. In conclusion, the present study indicated that the novel CDA produced by B. aryabhattai TCI-16 might be a promising option for bioconversion of chitin to the value-added chitosan.

Objective To explore the clinical features and genetic characteristics of Charcot-Marie-Tooth disease type 4B2 (CMT4B2) patients. Methods MTMR13/SBF2 gene mutations were screened by target region capture sequencing among a CMT4B2 Chinese family which included 3 patients. Results Case 1 (the proband) showed weakness in both lower limbs 6 years ago, running significantly slower than her classmates. The symptoms then became gradually worsened. Then the patient suffered from amyotrophy of thenar and hypothenar eminences and interosseus of her hands, bilateral finger joints could not be straightened, atrophy of bilateral leg muscles, talipes equinovarus appearance, and weakened tendon reflexes of limbs. Case 2 (the proband's younger brother) had weakness in both lower limbs and walked unsteadily 2 years ago, with running and going upstairs significantly slower than before. His abnormal walking gait got worsening in recent and he could not bend his feet upward in the stand or on the walk. He also presented amyotrophy of thenar and hypothenar eminences of his hands, mild atrophy of bilateral leg muscles, talipes equinovarus appearance, and weakened tendon reflex of limbs. Case 3 (the proband's another younger brother) also had difficulty in walking with the heel, and thenar and hypothenar eminences of his hands were mildly atrophic and the tendon reflexes were weakened. The result of proband's MTMR13/SBF2 gene test showed c.230G > A (p.Gln77Arg) and c.1537C > T (p.Gln513*) compound heterozygous mutations, her father carried c.230G > A (p.Gln77Arg) heterozygous mutation and her mother carried c.1537C > T (p.Gln513*) heterozygous mutation. Case 2 and Case 3 had the same compund heterozygous mutation as the proband. The 3 patients were diagnosed as CMT4B2, and their family was diagnosed as CMT4B2 pedigree. All patients were treated by mecobalamine. The proband was also treated by plaster immobilization to correct her talipes equinovarus. Conclusions CMT4B2 is a very rare and serious progressive type of CMT. Since there is no effective treatment of CMT4B2, we should carry out gene test as early as possible to make a clear diagnosis on patients and also take hereditary inquiry on the patient's family. For disease-causing gene carriers who want to have a baby, prenatal genetic diagnosis should be done to avoid the birth of CMT4B2 baby. For carriers of the gene without clinical symptoms or in early stage, close follow-up and active treatment should be taken to delay the onset and prevent talipes equinovarus or scoliosis, so as to improve the life quality of patients.

Objective To summarize the characteristics of fatty infiltration and edema of muscle MRI in Duchenne muscular dystrophy (DMD) patients. Methods A total of 70 DMD patients underwent Clinical Functional Scale and muscle MRI of the pelvic (gluteus maximus, gluteus medius, gluteus minimus, iliopsoas, piriformis, obturator internus, obturator externus, tensor fasciae latae, pectineus, erector spinae, psoas major muscle, iliacus), thigh (adductor magnus, gracilis, adductor longus, sartorius, rectus femoris, vastus intermedius, vastus medialis, vastus lateralis, biceps femoris, semitendinosus, semimembranosus) and leg (gastrocnemius, soleus, peroneus longus, tibialis posterior, tibialis anterior, flexor hallucis longus, extensor hallucis longus, extensor digitorum longus). T1WI fatty infiltration grade and fat suppression T2WI edema grade were performed to analyze the imaging features of fatty infiltration and edema. Results In 31 muscles, 30 muscles (96.77% ) presented fatty infiltration and 23 (74.19% ) presented edema. The occurrence rate of fatty infiltration was higher than that of edema. A total of 21 muscles including gluteus maximus, were found T1WI fatty infiltration grades greater than 2, and 10 muscles including tibialis posterior, were less than 1. Five muscles including soleus were found fat suppression T2WI edema grade 3. Spearman rank correlation analysis showed positive correlations between T1WI fatty infiltration grade and Clinical Functional Scale in 7 pelvic muscles [gluteus maximus (rs = 0.518, P = 0.016), gluteus medius (rs = 0.528, P = 0.014), gluteus minimus (rs = 0.528, P = 0.014), iliopsoas (rs = 0.695, P = 0.000), piriformis (rs = 0.451, P = 0.040), pectineus (rs = 0.567, P = 0.009), erector spinae (rs = 0.499, P = 0.025)], 7 thigh muscles [adductor magnus (rs = 0.607, P = 0.005), adductor longus (rs = 0.547, P = 0.013), rectus femoris (rs = 0.614, P = 0.004), vastus intermedius (rs = 0.566, P = 0.009), vastus medialis (rs = 0.522, P = 0.018), vastus lateralis (rs = 0.503, P = 0.024), biceps femoris (rs = 0.508, P = 0.022)] and 3 leg muscles [gastrocnemius (rs = 0.715, P = 0.001), peroneus longus (rs = 0.571, P = 0.017), tibialis posterior (rs = 0.514, P = 0.035)]. There was no correlation between T1WI fatty infiltration grade and Clinical Functional Scale in other muscles (P > 0.05, for all). There was no correlation between fat suppression T2WI edema grade and Clinical Functional Scale in all muscles (P > 0.05, for all). Conclusions Fatty infiltration grade can be well used to assess the severity of DMD. The role of muscle edema on assessing clinical severity should be analyzed according to individual muscle characteristics. DOI: 10.3969/j.issn.1672-6731.2018.07.006

Objective To study the characteristics and diagnosis of limb-girdle muscular dystrophy type 2D (LGMD2D). Methods The clinical characteristics, EMG, muscle MRI and muscle pathological studies of 2 female patients in a family with LGMD2D were analyzed. Genetic analysis was used in the diagnosis of this disease. The cases were reported along with related literatures review. Results The onset of the proband and her younger sister occurred at 3 years old with progressive proximal muscle weakness of four limbs as the main clinical manifestation. The serum creatine kinase (CK) was significantly high (> 50 × 10 3 U/L). EMG showed myogenic damage. Muscle MRI indicated partial muscle atrophy, fatness or fiber edema. Muscle pathological examination of the proband's younger sister revealed skeletal muscle necrosis and focal regeneration, partial striated muscle disappearance, and the muscle fibers in different sizes. Sequencing of all 10 coding exons of the SGCA gene in 2 patients revealed the same mutation: a c.262delT (p.Phe88SerfsX123) frameshift mutation in exon 3 and a c.409G > A (p.Glu137Lys) missense mutation in exon 5. Their mother was a carrier of SGCA gene c.409G > A (p.Glu137Lys) mutation. c.409G > A (p.Glu137Lys) is a mutation already found, and c.262delT (p.Phe88SerfsX123) is a novel mutation. The proband's father did not take the genetic test for some reason. Conclusions In case of a female with Duchenne muscular dystrophy (DMD).like symptom, if she has been excluded from the DMD gene carrier, pedigree analysis and genetic analysis involving limb . girdle muscular dystrophy (LGMD) should be conducted to facilitate the diagnosis of the LGMD and its subtypes. DOI: 10.3969/j.issn.1672-6731.2017.08.010

Adipose-derived stem cells have been shown to promote peripheral nerve regeneration through the paracrine secretion of neurotrophic factors. However, it is unclear whether these cells can promote myogenic differentiation in muscular dystrophy. Adipose-derived stem cells (6 × 10 6 ) were injected into the gastrocnemius muscle of mdx mice at various sites. Dystrophin expression was found in the muscle fibers. Phosphorylation levels of Akt, mammalian target of rapamycin (mTOR), eIF-4E binding protein 1 and S6 kinase 1 were increased, and the Akt/mTOR pathway was activated. Simultaneously, myogenin levels were increased, whereas cleaved caspase 3 and vimentin levels were decreased. Necrosis and fibrosis were reduced in the muscle fibers. These findings suggest that adipose-derived stem cells promote the regeneration and survival of muscle cells by inhibiting apoptosis and fibrosis, thereby alleviating muscle damage in muscular dystrophy.

Background DMD gene point mutation, mainly nonsense mutation, always cause the most severe Duchenne muscular dystrophy (DMD). However, we also observed some cases of Becker muscular dystrophy (BMD) carrying DMD point mutation. This paper aims to explore the mechanism of DMD point mutation causing BMD, in order to enhance the understanding of mutation types of BMD. Methods Sequence analysis was performed in 11 cases of BMD confirmed by typical clinical manifestations and muscle biopsy. The exon of DMD gene was detected non-deletion or duplication by multiplex ligation-dependent probe amplification (MLPA). Results Eleven patients carried 10 mutation types without mutational hotspot. Six patients carried nonsense mutations [c.5002G>T, p.(Glu1668X); c.1615C > T, p.(Arg539X); c.7105G > T, p.(Glu2369X); c.5287C > T, p.(Arg1763X); c.9284T > G, p.(Leu3095X)]. One patient carried missense mutation [c.5234G > A, p.(Arg1745His)]. Two patients carried frameshift mutations (c.10231dupT, c.10491delC). Two patients carried splicing site mutations (c.4518 + 3A > T, c.649 + 2T > C). Conclusions DMD gene point mutation may result in BMD with mild clinical symptoms. When clinical manifestations suggest the possibility of BMD and MLPA reveals non?deletion or duplication mutation of DMD gene, BMD should be considered. Study on the mechanism of DMD point mutation causing BMD is very important for gene therapy of DMD. DOI: 10.3969/j.issn.1672-6731.2015.06.005

Objective Use T2 mapping to evaluate the fatty infiltration of thigh muscles in Duchenne muscular dystrophy (DMD) patients, so as to analyze the value of T2 mapping and T2 relaxation time in the diagnosis of DMD. Methods Sixteen DMD patients who were admitted from January 2004 to January 2013 in our hospital and were diagnosed by clinical confirmation and gene detection have participated into this study. These 16 male patients formed the DMD group. Six age- and sex-matched healthy boys were selected as control group. Clinical functional scale, thigh axial T1WI-turbo spin echo (TSE), T2WI-TSE, spectral attenuated inversion recovery (SPAIR)-T2WI and T2 mapping were performed in both 2 groups. T1WI fatty infiltration scale and T2 relaxation time were assessed in adductor magnus, gracilis, adductor longus, sartorius, rectus femoris, vastus intermedius, vastus medialis, vastus lateralis, biceps femoris, semitendinosus and semimembranosus. Spearman rank correlation was conducted to assess the correlation between T2 relaxation time and T1WI fatty infiltration scale or clinical functional scale. Results Compared with control group, the T2 relaxation time of 8 muscles (adductor magnus, adductor longus, rectus femoris, vastus intermedius, vastus medialis, vastus lateralis, biceps femoris and semimembranosus) in DMD group were prolonged (P < 0.05, for all). The longest average T2 relaxation time was found in adductor magnus. The T2 relaxation time of adductor magnus, vastus intermedius, vastus lateralis, biceps femoris, rectus femoris, adductor longus and vastus medialis was positively correlated with T1WI fatty infiltration scale (P < 0.05, for all), and the T2 relaxation time of adductor magnus and semimembranosus was positively correlated with clinical funetional scale (P < 0.05, for all). A positive correlation was found in adductor magnus between T2 relaxation time and both T1WI fatty infiltration scale (rs = 0.867, P = 0.000) and clinical functional scale (rs = 0.651, P = 0.005). Conclusions T2 relaxation time could be used in the quantitative and objective analysis of DMD clinical severity, and adductor magnus was considered to be the most valuable muscle to reflect the clinical severity of DMD. DOI: 10.3969/j.issn.1672-6731.2015.06.004

Objective The clinical manifestation and electron transfer flavoprotein dehydrogenase (ETFDH) gene mutation of riboflavin-responsive lipid storage myopathy were analyzed for early diagnosis and treatment.mutation of riboflavin-responsive lipid storage myopathy were analyzed for early diagnosis and treatment. Methods Clinical material, ETFDH gene mutation and the motor function before and after vitamin B2 treatment in 3 patients from 2 predigrees were collected from August 2012 to March 2013 in our hospital. Results Case 1 was 16-year-old female. The chief complaint was difficulty of breathing and expectorating for over 3 years. Clinical symptoms included progressive respiratory muscle and proximal limb muscle weakness and worsen by fever, cardiac involvement, myopathic electromyography (EMG) changes and deposition of lipid droplets in muscle fiber by oil red O staining. Case 2 and Case 3 were brothers, the chief complaint of whom was fatigue after exercise for more than 1 year and 1 month, respectively. Clinical symptoms included significantly weakness of lower limbs and neck muscles after exercise and myopathic EMG changes. All 3 patients from two predigrees presented ETFDH gene mutation [c.250G > A (Ala84Thr) homozygous mutations and c.250G > A (Ala84Thr) and c.524G > A (Arg175His) compound heterozygous mutations, respectively]. They all had a dramatic response to vitamin B2 treatment with muscle strength and motor function recovering to normal. The symptoms of Case 1 were completely disappeared with vitamin B2 treatment for over 10 months, including respiratory muscle and proximal limb muscle weakness, and the motor function of her limbs returned to normal, characterized by completing over 10 squat-stand in 1 min. Case 2 could walk and run as ordinary people, raise his head without difficulty and play basketball about 2 h without fatigue after vitamin B2 treatment for over 2 months. Case 3 could participate in any kind of strenuous exercise without fatigue after vitamin B2 treatment for over 2 months. Conclusions Riboflavin-responsive lipid storage myopathy is mainly characterized by proximal limb and trunk muscle weakness and intolerance of movement, however, rare cases with first symptom of respiratory muscle weakness should also be concerned. In addition, it is a treatable genetic disease. The patients could be cured or significantly improved with vitamin B2 monotherapy. So vitamin B2 exploratory treatment should be given when the patients are suspected of riboflavin-responsive lipid storage myopathy. doi: 10.3969/j.issn.1672-6731.2014.06.004

Objective In order to make a well understanding on glycogen storage disease typeⅡ (GSDⅡ), this paper explored clinical features and genetic analysis of 7 patients with late-onset glycogen storage disease typeⅡ. Methods Clinical data of 7 patients with late-onset glycogen storage disease type Ⅱ were collected and acid α-glucosidase (GAA) gene sequencing was performed. Results Seven patients who belong to 4 families were at the age of 13-31 years old. The first symptom occurred at 6-17 years old, and the age at first and definitive diagnosis was 12-29 and 12-30 years old, respectively. The initial symptoms were mostly related to limb girdle muscular atrophy and weakness. The GAA activity ranged from 0 to 5.27 nmol/(mg·h). Sequencing analysis revealed 14 sequence variants, including 2 novel mutations (Q81X and c.1355_1356delC), 2 pseudodeficiency alleles (G576S and E689K), 8 polymorphic loci, and 2 sequence variants previously related with glycogen storage disease type Ⅱ pathogenesis (W746C and D645E). Conclusions Due to the apparently diagnostic delay, prognosis of patients with glycogen storage disease type Ⅱ could be improved by increasing the clinician's awareness of the disease. It is essential to combine clinical history with GAA activity and GAA gene analysis when we make a definitive diagnosis of glycogen storage disease type Ⅱ. Though siblings share the same set of GAA mutations, the phenotype regarding the course and severity of disease could vary substantially. doi: 10.3969/j.issn.1672-6731.2014.05.008