A marine lipopeptides-producing Bacillus amyloliquefaciens HY2-1 with a broad-spectrum antifungal and antibacterial activity and its fermentation kinetics study.

The antagonistic Bacillus amyloliquefaciens HY2-1 was a marine microbiology that was isolated previously from the seabed silt of Beibu Gulf in China by dual culture with Penicillium digitatum. As a continuous study, the present work focused on evaluating the antimicrobial activity, identifying the produced active components, and revealing the fermentation characteristics of B. amyloliquefaciens HY2-1, respectively. It was found that B. amyloliquefaciens HY2-1 exhibited a broad-spectrum antimicrobial activity against the tested seven phytopathogenic fungi and five pathogenic bacteria by producing Bacillus lipopeptides such as fengycin A (C14 to C19 homologues) and surfactin (C14 and C15 homologues). Morphological observation of P. digitatum under light microscope, scanning electron microscopy, transmission electron microscopy, and fluorescence microscope inferred that B. amyloliquefaciens exerted the antagonistic activity by damaging the fungal cell membrane, thus inhibiting the mycelium growth and sporification of phytopathogenic fungi. As a marine microbiology, our results showed that B. amyloliquefaciens could survive and metabolize even at the culture condition with 110 g/L of NaCl concentration, and the produced antimicrobial compounds exhibited excellent thermostability and acid-alkali tolerance. The dynamic models were further constructed to theoretically analyze the fermentation process of B. amyloliquefaciens HY2-1, suggesting that the synthesis of antimicrobial compounds was coupled with both cell growth and cell biomass. In conclusion, the marine lipopeptides-producing B. amyloliquefaciens HY2-1 showed a promising prospect to be explored as a biocontrol agent for plant disease control of crops and postharvest preservation of fruits and vegetables, especially due to its outstanding stress resistance and the broad-spectrum and effective antagonist on various phytopathogenic fungi.

Isolation, characterization, and genome sequencing of a novel chitin deacetylase producing Bacillus aryabhattai TCI-16.

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.

Adipose-derived stem cells enhance myogenic differentiation in the mdx mouse model of muscular dystrophy via paracrine signaling.

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 × 106) 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.

Diffusion-Tensor Imaging of Thigh Muscles in Duchenne Muscular Dystrophy: Correlation of Apparent Diffusion Coefficient and Fractional Anisotropy Values With Fatty Infiltration.

OBJECTIVE: The purpose of this study is to investigate the correlation of apparent diffusion coefficient (ADC) and fractional anisotropy (FA) values with fatty infiltration in the thigh muscles of patients with Duchenne muscular dystrophy (DMD) using diffusion-tensor imaging (DTI). SUBJECTS AND METHODS: Twenty-one boys with DMD were recruited. The grade of fatty infiltration and the ADC and FA values of four thigh muscles (rectus femoris, semitendinosus, sartorius, and gracilis) were measured, and the FA and ADC values were compared with the grade of fatty infiltration. Twenty age-matched healthy boys were enrolled as the control group. The differences in the ADC and FA values of the thigh muscles between patients with DMD and the control group were compared. RESULTS: The patients with DMD showed lower FA values and higher ADC values in all measured muscles when compared with the control group. The FA and ADC values were correlated with the grade of fatty infiltration. For the rectus femoris muscle, r = -0.753 and p = 0.007 for FA, and r = 0.685 and p = 0.001 for ADC. For the semitendinosus muscle, r = -0.621 and p = 0.041 for FA, and r = 0.705 and p = 0.021 for ADC. For the sartorius muscle, r = -0.662 and p = 0.027 for FA, and r = 0.701 and p = 0.017 for ADC. For the gracilis muscle, r = -0.618 and p = 0.043 for FA, and r = 0.695 and p = 0.022 for ADC. CONCLUSION: Damage to the thigh muscles in patients with DMD can be detected by ADC and FA values using DTI. DTI can be used to assess the severity of the disease.

[Study on Duchenne muscular dystrophy gene mutation and prenatal diagnosis].

OBJECTIVE: To explore the characteristics of DNA mutations underlying Duchenne muscular dystrophy and provide prenatal diagnosis. METHODS: Multiplex ligation-dependent probe amplification (MLPA) and denaturing high performance liquid chromatography (DHPLC) were applied for analyzing DMD gene mutations in 388 unrelated Chinese patients and 53 fetuses. RESULTS: Respectively, 230 and 43 subjects were found to harbor a deletion (59.28%) or duplication (11.08%). Two deletion hotspots were identified, which have located at exons 45-54 and exons 3-19. Duplications were mainly detected at exons 2-43. Point mutations were identified in 29.64% of patients. Fifty three fetuses were prenatal diagnosed, among which 18 were identified as patients. CONCLUSION: Frequencies of DMD gene deletions and duplications in China are similar to global data. Prenatal diagnosis can help to reduce births of DMD patients.

[Correlation between genotypes and phenotypes in pseudohypertrophic muscular dystrophy].

OBJECTIVE: To explore the correlation between genotypes and phenotypes in Chinese patients with pseudohypertrophic muscular dystrophy. METHODS: Patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) were diagnosed clinically. Multiplex ligation-dependent probe amplification (MLPA) were performed to detect potential DMD gene mutations. The results were analyzed statistically. RESULTS: Among 280 patients, 238(85.0%) were diagnosed with DMD, 35(12.50%) were diagnosed with BMD and 7(2.5%) were diagnosed with intermediate muscular dystrophin (IMD). Among these, 252(92.31%) were in-frame mutations, and 21(7.69%) were out-of-frame mutations. Twelve patients with DMD have carried in-frame mutations, 9 with BMD have carried frame-shift mutations, and 7 IMD patients have carried frame-shift mutation. CONCLUSION: Most of the genotypes and phenotypes of DMD have complied with the reading-frame hypothesis. Patients with BMD with frame-shift mutations may facilitate understanding of the pathogenesis of DMD, and provide a theoretical basis for clinical therapy.

Restoration of muscle fibers and satellite cells after isogenic MSC transplantation with microdystrophin gene delivery.

Duchenne muscular dystrophy is the most prevalent inheritable muscle disease. Transplantation of autologous stem cells with gene direction is an ideal therapeutic approach for the disease. The current study aimed to investigate the restoration of myofibers in mdx mice after mdx bone marrow-derived mesenchymal stem cell (mMSC) transplantation with human microdystrophin delivery. Possible mechanisms of action were also studied. In our research, mMSCs were successfully transduced by retrovirus carrying a functional human microdystrophin gene. Transplantation of transduced mMSCs enabled persistent dystrophin restoration in the skeletal muscle of mdx mice up to the 12th week after transplantation. Simultaneous coexpression of human microdystrophin and desmin showed that implanted mMSCs are capable of long-term survival as muscle satellite cells.

[Values of serum copper and serum free copper in the diagnosis and monitoring of Wilson's disease and its carriers].

OBJECTIVE: To explore the values of serum copper and serum free copper in the diagnosis of Wilson's disease (WD), its carrier and viral hepatitis and explore the guiding significance of monitoring serum copper in the treatment of WD. METHODS: A total of 80 WD patients (hepatic type, n = 60; encephalic type, n = 20), 30 carriers, 20 patients with viral hepatitis were enrolled and their levels of serum copper were determined. The neural symptoms were scored by modified Young grade. Hemogram, hepatic functions, blood clotting functions, serum copper and urinary copper were tested throughout all 8 courses of treatment with sodium dimercaptopropane sulfonate (DMPS). The patients were treated with zinc after discharging. All data were analyzed. RESULTS: The free serum copper increased in the patients with WD (0.17 mg/L ± 0.04 mg/L), carriers (0.13 mg/L ± 0.03 mg/L) and severe viral hepatitis (0.12 mg/L). A slight increase was also observed in the WD carriers. The level of serum copper was correlated with hepatic functions but not with the severity of neural symptoms. The serum copper increased in the patients with no improvement of neural symptoms. However, the serum copper decreased in the WD patients with the improvement of neural symptoms. The serum copper was stabilized at approximately 0.2 mg/L during the long-term treatment period. CONCLUSION: There is auxiliary diagnosis significance of serum copper in the determination of WD. Hepatic functions in hepatic type WD affect the level of serum copper. The serum copper of encephalic type WD can not indicate the severity of neural symptoms. The elevated level of serum copper indicates a poor prognosis. The serum copper is an effective marker in monitoring the development and therapeutic efficacy of the disease.

[Preliminary study of the spatial structural and functional changes of dystrophin after exon-3 deletion].

OBJECTIVE: To explore the structural and functional changes of dystrophin molecule after exon 3 deletion. METHODS: Three-dimensional models of dystrophin comprising the major domains were established before and after exon 3 deletion using SWISS-MODEL server. The motifs and structural domains of dystrophin after exon 3 deletion were searched in Pfam database, and the crystal structure of the actin-binding domain in the dystrophin molecule was analyzed using Rasmol software. RESULTS: Torsion of the N-terminal actin-binding domain occurred in the dystrophin molecule after deletion of exon 3. Homology analysis based on Pfam database searches indicated that following exon 3 deletion, the Bit score of the first calponin homology (CH1) domain was decreased from 108 to 36.5 while its expectation value increased from 2.3e-9 to 8.1e-8. The deletion also resulted in the absence of the spiral region C from the CH1 domain. CONCLUSION: Exon 3 deletion in the dystrophin-coding sequence decreases the stability of CH1 domain and prevents the formation of the junction interface where dystrophin binds to actin. The bioinformatics approach provides a new alternative for investigation of the pathogenesis of DMD pathogenesy investigation.