Melanin biopolymer synthesis using a new melanogenic strain of Flavobacterium kingsejongi and a recombinant strain of Escherichia coli expressing 4-hydroxyphenylpyruvate dioxygenase from F. kingsejongi.

BACKGROUND: Melanins are a heterologous group of biopolymeric pigments synthesized by diverse prokaryotes and eukaryotes and are widely utilized as bioactive materials and functional polymers in the biotechnology industry. Here, we report the high-level melanin production using a new melanogenic Flavobacterium kingsejongi strain and a recombinant Escherichia coli overexpressing F. kingsejongi 4-hydroxyphenylpyruvate dioxygenase (HPPD). RESULTS: Melanin synthesis of F. kingsejongi strain was confirmed via melanin synthesis inhibition test, melanin solubility test, genome analysis, and structural analysis of purified melanin from both wild-type F. kingsejongi and recombinant E. coli expressing F. kingsejongi HPPD. The activity of F. kingsejongi HPPD was demonstrated via in vitro assays with 6 × His-tagged and native forms of HPPD. The specific activity of F. kingsejongi HPPD was 1.2 ± 0.03 µmol homogentisate/min/mg-protein. Bioreactor fermentation of F. kingsejongi produced a large amount of melanin with a titer of 6.07 ± 0.32 g/L, a conversion yield of 60% (0.6 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.03 g/L·h, indicating its potential for industrial melanin production. Additionally, bioreactor fermentation of recombinant E. coli expressing F. kingsejongi HPPD produced melanin at a titer of 3.76 ± 0.30 g/L, a conversion yield of 38% (0.38 ± 0.03 g melanin per gram tyrosine), and a productivity of 0.04 g/L·h. CONCLUSIONS: Both strains showed sufficiently high fermentation capability to indicate their potential as platform strains for large-scale bacterial melanin production. Furthermore, F. kingsejongi strain could serve as a model to elucidate the regulation of melanin biosynthesis pathway and its networks with other cellular pathways, and to understand the cellular responses of melanin-producing bacteria to environmental changes, including nutrient starvation and other stresses.

Implantation of polymer scaffolds seeded with neural stem cells in a canine spinal cord injury model.

BACKGROUND AIMS: Combinatorial approaches employing diverse therapeutic modalities are required for clinically relevant repair of injured spinal cord in human patients. Before translation into the clinic, the feasibility and therapeutic potential of such combinatorial strategies in larger animal species need to be examined. METHODS: The present study tested the feasibility of implanting polymer scaffolds via neural stem cell (NSC) delivery in a canine spinal cord injury (SCI) model. The poly(lactic-co-glycolic acid) (PLGA) scaffolds seeded with human NSC were implanted into hemisected canine spinal cord. RESULTS: The PLGA scaffolds bridged tissue defects and were nicely integrated with residual canine spinal cord tissue. Grafted NSC survived the implantation procedure and showed migratory behavior to residual spinal cord tissue. Ectopic expression of a therapeutic neurotrophin-3 gene was also possible in NSC seeded within the PLGA scaffolds. CONCLUSIONS: Our description of a canine SCI model would be a valuable resource for pre-clinical trials of combinatorial strategies in larger animal models.

Therapeutic effects of a liquid bandage prepared with cellulose powders from Styela clava tunics and Broussonetia kazinoki bark: Healing of surgical wounds on the skin of Sprague Dawley rats.

Cellulose in different forms has extensively been applied in biomedical treatments, including scaffolding, tissue engineering and tissue formation. To evaluate the therapeutic effects of a liquid bandage (LB) prepared with cellulose powders from Styela clava tunics (SCT) and Broussonetia kazinoki bark (BSLB) for healing cutaneous wounds, the remedial effects of a low concentration (LoBSLB) and a high concentration (HiBSLB) of BSLB on skin regeneration and toxicity in Sprague Dawley rats. Results indicated that the total area of skin involved in the surgical wound was lower in the BSLB­treated group compared with the Vehicle­treated group at days 4­12, although some variations were observed in the HiBSLB­treated group. In addition, the BSLB­treated group showed significantly enhanced width of the re­epithelialization region and epidermal thickness when compared with the Vehicle­treated group. Furthermore, significant stimulation in the expression level of collagen­1 and the signaling pathway of VEGF after topical application of BSLB was indicated. No liver or kidney toxicities were detected for either doses of BSLB. Overall, the results of the present study suggest that BSLB accelerates the process of wound healing in surgical skin wounds of Sprague Dawley rats through stimulation of re­epithelialization and connective tissue formation, without any accompanying significant toxicity.

Development of polyvinyl alcohol-sodium alginate gel-matrix-based wound dressing system containing nitrofurazone.

Polyvinyl alcohol (PVA)/sodium alginate (SA) hydrogel matrix-based wound dressing systems containing nitrofurazone (NFZ), a topical anti-infective drug, were developed using freeze-thawing method. Aqueous solutions of nitrofurazone and PVA/SA mixtures in different weight ratios were mixed homogeneously, placed in petri dishes, freezed at -20 degrees C for 18h and thawed at room temperature for 6h, for three consecutive cycles, and evaluated for swelling ratio, tensile strength, elongation and thermal stability of the hydrogel. Furthermore, the drug release from this nitrofurazone-loaded hydrogel, in vitro protein adsorption test and in vivo wound healing observations in rats were performed. Increased SA concentration decreased the gelation%, maximum strength and break elongation, but it resulted into an increment in the swelling ability, elasticity and thermal stability of hydrogel film. However, SA had insignificant effect on the release of nitrofurazone. The amounts of proteins adsorbed on hydrogel were increased with increasing sodium alginate ratio, indicating the reduced blood compatibility. In vivo experiments showed that this hydrogel improved the healing rate of artificial wounds in rats. Thus, PVA/SA hydrogel matrix based wound dressing systems containing nitrofurazone could be a novel approach in wound care.

Salivary cortisol and DHEA levels in the Korean population: age-related differences, diurnal rhythm, and correlations with serum levels.

PURPOSE: The primary objective of this study was to examine the changes of basal cortisol and DHEA levels present in saliva and serum with age, and to determine the correlation coefficients of steroid concentrations between saliva and serum. The secondary objective was to obtain a standard diurnal rhythm of salivary cortisol and DHEA in the Korean population. MATERIALS AND METHODS: For the first objective, saliva and blood samples were collected between 10 and 11 AM from 359 volunteers ranging from 21 to 69 years old (167 men and 192 women). For the second objective, four saliva samples (post-awakening, 11 AM, 4 PM, and bedtime) were collected throughout a day from 78 volunteers (42 women and 36 men) ranging from 20 to 40 years old. Cortisol and DHEA levels were measured using a radioimmunoassay (RIA). RESULTS: The morning cortisol and DHEA levels, and the age- related steroid decline patterns were similar in both genders. Serum cortisol levels significantly decreased around forty years of age (p < 0.001, when compared with people in their 20s), and linear regression analysis with age showed a significant declining pattern (slope=-2.29, t=-4.297, p < 0.001). However, salivary cortisol levels did not change significantly with age, but showed a tendency towards decline (slope=-0.0078, t=-0.389, p=0.697). The relative cortisol ratio of serum to saliva was 3.4-4.5% and the ratio increased with age (slope=0.051, t=3.61, p < 0.001). DHEA levels also declined with age in saliva (slope=-0.007, t=-3.76, p < 0.001) and serum (slope=-0.197 t=-4.88, p < 0.001). In particular, DHEA levels in saliva and serum did not start to significantly decrease until ages in the 40s, but then decreased significantly further at ages in the 50s (p < 0.001, when compared with the 40s age group) and 60s (p < 0.001, when compared with the 50 age group). The relative DHEA ratio of serum to saliva was similar throughout the ages examined (slop=0.0016, t=0.344, p=0.73). On the other hand, cortisol and DHEA levels in saliva reflected well those in serum (r=0.59 and 0.86, respectively, p < 0.001). The highest salivary cortisol levels appeared just after awakening (about two fold higher than the 11 AM level), decreased throughout the day, and reached the lowest levels at bedtime (p < 0.001, when compared with PM cortisol levels). The highest salivary DHEA levels also appeared after awakening (about 1.5 fold higher than the 11 AM level) and decreased by 11 AM (p < 0.001). DHEA levels did not decrease further until bedtime (p=0.11, when compared with PM DHEA levels). CONCLUSION: This study showed that cortisol and DHEA levels change with age and that the negative slope of DHEA was steeper than that of cortisol in saliva and serum. As the cortisol and DHEA levels in saliva reflected those in serum, the measurement of steroid levels in saliva provide a useful and practical tool to evaluate adrenal functions, which are essential for clinical diagnosis.

A Case of Infective Endocarditis caused by Abiotrophia defectiva in Korea.

Abiotrophia defectiva, a nutritionally variant streptococci can cause bacteremia, brain abscess, septic arthritis and in rare cases, infective endocarditis, which accounts for 5-6% of all cases. A. defectiva is characteristically difficult to diagnose and the mortality, morbidity and complication rates are high. Here, we discuss a case of infective endocarditis caused by A. defectiva. A 62-year-old female had previously undergone prosthetic valve replacement 6 years prior to admission. She developed infective endocarditis after tooth extraction. Her endocarditis was successfully treated with antimicrobial therapy and mitral valve replacement surgery. This is the first case of infective endocarditis caused by A. defectiva reported in Korea. This case shows that A. defectiva could be considered as a causative organism of infective endocarditis in Korea.

Development of clindamycin-loaded wound dressing with polyvinyl alcohol and sodium alginate.

To develop a clindamycin-loaded wound dressing, cross-linked hydrogel films were prepared using freeze-thawing method with various mixtures of polyvinyl alcohol (PVA) and sodium alginate (SA). The physicochemical properties such as swelling ratio, tensile strength and elongation of hydrogels were evaluated. The drug release from this clindamycin-loaded hydrogel, in vitro protein adsorption test and in vivo wound healing observations in rats were then performed. Increased SA concentration decreased the gelation %, maximum strength and break elongation, but it resulted into an increment in the swelling ability, elasticity and thermal stability of hydrogel film. However, SA had insignificant effect on the release of clindamycin. This hydrogel improved the healing rate of artificial wounds in rats. Thus, a clindamycin-loaded wound dressing with PVA and SA hydrogel should be a candidate for wound care.