Earticle

For achieving biodiesel production from microalgae, high density cultivation of microalgae is the most important step through whole process. Microalgal cultivation has been limited by the fact that there is a hurdle to efficiently or economically provide the light energy, which is the most expensive and essential element for microalgal growth, especially in a photobioreactor (PBR). In this study, we employed the concept of flashing illumination, which should in turn reduce the cost of microalgal cultivation significantly.The effects of inoculum size, light intensity, and duration of light or dark on growth rate and lipid content of Chlamydomonas reinhardtii, one of the model microalgae, were investigated under flashing illumination. This research showed that microalgal cultivation under flashing illumination is meaningful and promising because it is capable of saving energy cost with similar performance compared with continuous illumination.

Nowadays, research effort is focused on looking for new and effective nutritional source and new progressive fermentation techniques enabling the achievement of both high substrate conversion and high production yields. In this study, bioethanol by-products were used as nutrients by Klebsiella oxytoca ATCC 43863 for the production of 2,3-butanediol (2,3-BDO), a promising bulk chemical which has potential applications in plastics, solvent, and anti-freeze solutions preparation. Bioethanol by-products were supplied from the commercial ethanol plant of Changhae Ethanol Co., Ltd, and solely used as fermentation medium without an addition of other ingredients. To evaluate the nutritional value of bioethanol by-products as fermentation medium, the total nitrogen, crude protein, and free amino acid were analysed and compared with a commercial yeast extract. K. oxytoca ATCC 43863 efficiently produced 2,3-BDO from these medium and increased the 2,3-BDO productivity and yield, compared to control medium containing 2 g/L yeast extract. Therefore, bioethanol by-products could be considered as an alternative fermentation medium for 2,3-BDO production.

Bacillus sp. strain BCNU 5020 was isolated from industrial wastewater in Korea. Strain BCNU 5020 was able to utilize dibenzothiophene, naphthalene, pentachlorophenol, phenanthrene, phenol and toluene as a sole source of carbon and energy under aerobic condition. Based on morphological, physiological and biochemical characteristics, and 16S rDNA sequence, strain BCNU 5020 was identified as Bacillus sp. The effect of the pH, temperature and concentration on the naphthalene degradation was studied through incubation with various kinds of condition. Biodegradation of naphthalene by strain BCNU 5020 was also confirmed by LC and LC-MS. Bacillus sp. BCNU 5020 was also determined to be tolerant to various kinds of organic solvents. Therefore, Bacillus sp. strain BCNU 5020 is of great practical significance both in bioremediation and biotreatment.(This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (Contract No 2010-0009141))

The 2,3-butanediol (2,3-BD) is a valuable chemical used as a solvent, liquid fuel, and precursor of many synthetic polymers and resins. Consequently, variety of researches have reported to achieve the efficient production of 2,3-BD (Zeng & Sabra, 2011) even though 2,3-BD producing level is not enough to apply industry field. Raoultella sp. B6 isolated from an oil-contaminated soil sample could produce 2,3-BD from various single carbon sources. The flask test was performed for the confirmation of 2,3-BD producing condition of the isolate such as initial pH, glucose concentration, and optimum temperature for bacterial growth and production of 2,3-BD. The pH condition and culture temperature were very critical in bacterial growth and 2,3-BD producing. Based on the batch test, fed-batch fermentation was performed to evaluate the potential 2,3-BD producing ability of the isolate under several pH condition. The initial pH 7.0 decreased to 5.5 during cell growth, and then maintained at 5.5 with 5N KOH. As a result, R. sp. B6 produced 2,3BD at highest concentration (64.7g/L), yield (0.55g/g) and productivity (0.54g/L/h). It suggests that the R. sp. B6 has potential industrial value for the 2,3-BD production through the establishment of optimum cultivative condition about variety of factors and engineering the bacterium.

A variety of toxic bloom-forming algae damaged marine ecosystems in the ocean. Recently, the viruses infecting the harmful algae were isolated and characterized. It was found that the viruses control the population dynamics by terminating the harmful algae. Previously, the HcRNAV strains infecting Heterocapsa circularisquama that causes HAB (Harmful algal blooming) were isolated and their intraspecies infectivity was discovered. The genome sequencing of the viruses identified a capsid gene and it was speculated that the variable regions in the capsid surfaces determined the specificities. To utilize the capsid as tool of HAB control, the expression and purification of the proteins were attempted in this study. To express the capsid protein from one of the HcRNAV strains, HcRNAV109, the gene encoding the protein was synthesized for codon optimization. The gene was cloned into pTXB1 expression vector containing intein tag gene and its expression was induced by IPTG in E.coli BL21(DE3). The recombinant protein was purified with chitin affinity chromatography column and its formation into the viral like protein (VLP) was confirmed by transmission electron microscopy (TEM). To ensure attachment of the recombinant capsid into the algae with host specificity, it was labeled with fluorescein isothiocyanate (FITC) during a chemical dissociation and reassociation process. The following infection experiment showed that the capsid of HcRNAV109 exhibited specific affinity to H. circularisquama strain HY9423.

Uptake [NiFe]-hydrogenase of Hydrogenovibrio marinus is O2-tolerant catalysis with an applicable potential for H2 production under aerobic condition. Here, genomic organization of the hydrogenase operons was analyzed from the genome sequence of H. marinus. Three gene clusters comprising hup and hyp genes are distributed over 33.8 kbp region, flanking a sulfite reductase operon. The organization is similar to that of Rhodobacter capsulatus, except for some variations. A gene that has no homology to any other genes was found in the downstream of hupC gene (coding for b-type cytochrome), which may have functional role for O2 resistance. The expression vector consisting of hyp and hup genes were constructed, aiming to heterologously express the hydrogenase having structural identity with the authentic enzyme. This study will provide information for the heterologous expression and the analysis of the distinguished [NiFe]-hydrogenase.

In an era of increasing oil prices and climate concerns, biodiesel has gained attention as alternative energy sources. Biodiesel derived from microalgae is a potential renewable and carbon neutral alternative to petroleum fuels. One of the most important decisions in obtaining oil from microalgae is the choice of algal species. A total of 32 microalgae cultures were isolated from an AMD (Acid Mine Drainage) in South Korea. 5 microalgae isolates were selected based on their morphology and ease of cultivation under our test conditions. The name of microalgae Nephroselmis sp. KGE 1, Nephroselmis sp. KGE 2, Uronema sp. KGE 8, Scenedesmus obiquus KGE 9 and Micractinium sp. KGE 10, based on 28S rRNA (D1D2 forward primer 5’-AGCGGAGGAAAAGAAACTA-3’,D1D2 revers primer 5’- TACTAGAAGGTTCGAT TA GT C-3’) sequence analyses. Nephroselmis sp. KGE 2 maximum growth of biomass concentration of 1.45±0.15 g dwt L-1 with a relatively lipid content of 20±0.5% w/w. Also, the fatty acids compositions of the studied species were mainly myristic, palmitic, palmitoleic, oleic, linoleic, g-linolenic, and linolenic acids. High concentration of oleic acid was also found in Nephroselmis sp. KGE 2, which led us to conclude that fatty acids of microalgae incubated in growth media were more effective for biodiesel production due to relatively lower saturation degree compared to the other cultures algal species. Our results suggest that Nephroselmis sp. KGE 2 can be a possible candidate species to produce oils for high quality biodiesel.

The redox balance in microbial metabolism is an important factor to determine end-products in the microbial fermentation. We supplied reducing power to a Clostridium strain to manipulate the redox metabolism using a cathode as a bioelectrochemical technique (1-3). The cyclic voltamogram showed a redox peak at -160 mV v.s. Ag/AgCl in the Clostridium culture grown in P2 medium with glucose as a carbon source. Electrochemically active Clostridium sp. was cultivated in a cathode compartment at an applied potential of -160 mV vs. Ag/AgCl. The electric current was consumed by the Clostridium without any mediator and it was increased to 1.5 mA according to the microbial growth. Interestingly, the production of butyrate and acetate from glucose was decreased in this bioelectrochemical system. However, butanol production (4 NADH consumed for 1 butanol) was increased up to 2.4 times. We measured the ratio of NADH/NAD+ within cells and found it was dependent on the current change. It was supposed that the cathode reduction of NAD+ induced more butanol production. This is the first report of controlling the cellular metabolism of the electroactive Clostridium given by an electrochemically introduced reducing equivalent.

An aerobic, chemoheterotrophic, mesophilic strain, designated CGM1-3EN was isolated from the enriched cultures of forest soil from Cheong-gye mountain, Korea. The 16S rRNA gene of the strain CGM1-3EN was sequenced and used as a query against EzTaxon database. The 16S rRNA gene sequence of the strain CGM1-3EN was aligned with the representative strains in a family Caulobacteraceae and manually curated. The phylogenetic analysis based on 16S rRNA gene analysis showed that the strain CGM1-3EN is included in a member of genus Asticcacaulis. The closest relatives of strain CGM1-3EN were Asticcacaulis taihuensis (96.38%) and Asticcacaulis benevestitus (96.31%). The phylogentic tree were inferred using four different tree-making algorithms including neighbour-joining, maximum-parsimony, UPGMA and maximum-likelyhood. The strain CGM1-3EN formed monophyletic group with A. taihuensis and A. benevestitus in all four different tree-making algorithms. Branch stability was assessed by 1,000 replicate neighbor-joining bootstrap replications and his monophyletic group was strongly supported by bootstrap value. The strain CGM1-3EN grew at up to 30 ℃.

Since yeast is often exposed to a number of environmental stresses including high ethanol concentration and temperature, the ability to grow and produce ethanol under such stressful conditions is an important factor in industrial bioethanol production. Trehalose is found in many organisms including Saccharomyces cerevisiae, and has been known to play an important role in enhancing various types of stress tolerance. In this study, Streptomyces albus trehalose-6-phosphate synthase gene (salC) was expressed in Saccharomyces cerevisiae, and the recombinant strain with salC gene showed significantly improved stress resistances and ethanol production. The stress sensitivity and viability tests indicated that the recombinant had a greater resistance to ethanol than the control. The results of flask cultures at an elevated temperature showed that the expression of salC played a good role of protecting cells from the heat stress. The recombinant strain was found to consume 100 g/L glucose and produce 39 g/L ethanol at 40 °C with an ethanol yield 6 % higher than that of the control strain. In the fed-batch experiment in a bioreactor to investigate ethanol production capability of the recombinant strain under a high-ethanol stress condition as in the industrial practice, it produced 69 g/L ethanol with about 18% higher yield and about 13% higher productivity than the control strain.

The reversibility of the electron transfer between electrodes and cells provides a promising way to produce sustainable transportation and chemical production needs. Typically, microbial electrosynthesis (MES) is conducted in a system, which consists of an anode, a cathode and a membrane separating the two. Microorganisms capture electrons derived from the anode to reduce carbon dioxide to multicarbon organic compounds. In this regard, MES is considered as a promising strategy for solving global environmental problem by reducing the greenhouse gas as well as energy depletion by converting electrical energy into forms that can be readily stored and distributed. However, such MES systems are limited by the difficult electrical wiring of microbes to the electrode. Here, we show the efficient wiring of microbes in a carbon nanofiber (CNF) matrix, which has extremely high surface area, excellent electrical conductivity and chemical inertness. The formation of carbon nanofiber-microbe composite biofilm facilitates the electron-transfer between electrode and microbes and accordingly improves the overall performance of MES. The effects of CNF on the MES systems of several acetogens were investigated.

The aim of this study is to find the optimum culture mode for Chlamydomonas reinhardtii among phototrophic, heterotrophic, and mixotrophic modes. In addition, the effect of different organic carbon sources (including acetate, glucose, glycerol, and sucrose) their influences the microalgal growth and oil production. Our results show that C. reinhardtii could grow under phototrophic, heterotrophic, and mixotrophic modes. C. reinhardtii exhibited superior growth in mixotrophic cultivation with all organic carbon sources compared to phototrophic and heterotrophic modes. Acetate was the optimum organic carbon source for mixotrophic cultivation of C. reinhardtii. Additionally, the feasibility of volatile fatty acids (VFAs) for microalgal growth and lipid accumulation was investigated.

With an increasing interest about microalgal based biodiesel production, many researches have been conducted to enhance a production yield and decrease a production cost. Among the whole steps of algal biodiesel producing process, the most expansive process is a cultivation. It needs land, facility, energy, water, temperature, chemicals, light and so on. It takes up 42% of total production cost.In this study, We use a plant hormone as an algal growth promoter. It can enhance the maximum cell density as well as lipid yield. Total cost of biomass and lipid production is analysed by the data based on cultivation conditions. The cost of plant hormone and growth medium is calculated by several references. Cost analysis of ACS grade chemical is based on Sigmaaldrich® and Gibco® and that of bulk chemical is based on Alibaba.com which is the one of the famous online trading platform and also the other website. Plant hormone is already wildly used in the agricultural industry for various purposes, so if we well adapt these chemical to microalgal cultivation, it will help the industrialization of microalgal biodiesel.

Jerusalem artichoke was exploiting alternative feedstock for fuel ethanol production. One of the low-requirement sugar crops, is an inexpensive and wildly available non-grain raw biomaterial containing cellulose and hemicelluloses in the stalk and a high content of inulin in the tuber. There are many advantages over grain crops: it can grow well in infertile land such as arid soil and salina, and resist many plant pests and diseases. And Kluyveromyces marxianus can inulinase and ethanol production at high temperature. In this study, Jerusalem artichoke stalk was pretreated by diluted acid treatment and then akali treatment. The optimum pretreatment conditions were at 121°C, 1 h retention time and 2% sulfuric acid solution and then 1M sodium hydroxide to dry biomass loading of 1:10 (w/w). The cellulose and hemicellulose from the tuber w ere released into the pretreatment. The sequentially acid/akali pretreatment biomass condisted of 51.6 % cellulose, 1.74 % hemicelluloses and 8.0 % lignin content afterward. Batch simultaneous saccharification and fermentation by Kluyveromyces maxianus, an inulinase activity yeast, was effectively performed and 29.0 g ethanol per 1 L of the fermented medium was yielded, the ethanol productivity was over 1.07 g ethanol/ g of dried Jerusalem artichoke and over 95% of total sugar was used by the yeast strain. This result showed that combining tuber to the stalk hydrolysate was a useful strategy of whole biomass utilization for effective fermentation of bioethanol production from Jerusalem artichoke.

Polylactide (PLA), which is one of the most important biodegradable and biocompatible polyesters that are derived from annually renewable biomass such as corn and sugar beets, has attracted much attention for its favorable material properties. The most efficient method for preparation of PLA is the ring-opening polymerization of the dimeric cyclic ester of lactic acid, lactide. Lactic acid is widely used in the food, pharmaceutical and cosmetic industries. It is also a major raw material for the production of polylactic acid (PLA) that is a biodegradable, environmentfriendly polymer which could be a substitute for synthetic plastics derived from petroleum feedstocks. Fermentative production of lactic acid offers the great advantage of producing optically pure L-or D- lactic acid depending upon the strains selected for fermentation. The optical purity of lactic acid is crucial for the physical properties of PLA. Though L-lactic acid can be polymerized to give crystalline (PLLA) suited to commercial uses, its application is limited by its low melting point. Complexing PLLA with poly D-lactic acid (PDLA), however, increases the melting point thus presenting an attractive solution to the heat sensitivity of PLA. However, fermentation of sugars to D-lactic acid is little studied and its microbial productivity is not well known. Therefore, we investigated D-lactic acid fermentation with a view to obtaining improved strains capable of producing D-lactic acid with enhanced productivities and finally we got a maximum lactic acid production 60 g/l. Fermentation based process requires maintenance of near neutral pH for high productivity and this necessitates addition of alkali in most of the cases. Alkali addition produces salt of lactic acid instead of lactic acid itself. To overcome this salt problem, we tested the electrodialysis based processes that do not require addition of acid or alkali again to convert lactate salts into lactic acid. Electrodialysis technology which is shown in Fig. 1 is based on electromigration of ions through a stack of cation and anion exchange membranes. Basically, it involves two steps. The first step called conventional electrodialysis (CED) separates and concentrates lactate salts. The second step called bipolar electrodialysis (BED) converts lactate salts into lactic acid. We adopted this two process and produced D-lactic aicd. Lactide is prepared by a two-step process: first, the lactic acid is converted into oligo (lactic acid) by a polycondensation reaction; second, the oligo(lactic acid) is thermally depolymerized to form the cyclic lactide via a unzipping mechanism. Through catalyst screening test for polycondensation and unzipping depolymerization reaction, we got a new method which shorten the whole reaction time as 50% level compared to the conventional method. Poly(L-lactide) was obtained from the ring-opening polymerization of L-lactide. We investigated various catalysts and polymerization conditions. Finally we got the best catalyst system and the scale-up technology.

Microalgae has received attention as an attractive feedstock for renewable biodiesel production due to their desirable characteristics such as rapid growth rate, high lipid contents, and resistance to contamination which can be utilized as microalgal bio-fuel. However, it is indispensable to further develop microalgae strains to display some ideal phenotype for more effective biofuel production. Toward this aim, targeted genetic manipulation was utilized to enhance lipid production in microalgae.When three kinds of microalgae were treated by MAP kinase inhibitor, the lipid content was increased in common. Therefore, MAP kinase affects the lipid biosynthesis in a negative way in microalgae. Based on this fact, repression of MAP kinase genes using RNA interference approach was employed in the model green alga Chlamydomonas reinhardtii. We currently obtain transformants and RT-PCR and phenotype analysis are going to be conducted to investigate the relationship between MAP kinase signaling and lipid biosynthesis. Regulation of MAP kinase genes allows the algal strains to contain high lipid contents for enhanced biofuel production.

Microalgae are a species that is receiving much attention as the hope of future energy source. Microalgae possess the ability to photosynthesize, producing half of the atmospheric oxygen and using the greenhouse gas carbon dioxide. The advantages of microalgae as a source of biodiesel include its high oil content, no use of agricultural land or products and biodegradability. However, they are disadvantageous in biodiesel production in that species with high lipid content grow slowly. And species that grow rapidly will accumulate little lipid. Therefore, in order to develop a strain that is more suitable for biodiesel production, genetic engineering is a pivotal step. For genetic improvement of Chlamydomonas reinhardtii, relatively simple mutagenesis method using a chemical mutagen Ethyl Methanesulfonate was adopted in this study. From a large pool of mutants generated, superior mutants were screened out. Since high-lipid content is the most essential characteristic of an ideal microalgae strain for biodiesel production, lipid-rich strains were sought. Then, their characteristics as the potential strain for biodiesel were further analyzed using Nile-red staining, photoluminescence and gas chromatography.

Megasphaera elsdenii NCIMB 702410 is a gram-negative strictly anaerobic bacterium isolated in the rumen of cattle and sheep. This strain is able to utilize sucrose to produce 4.53 g/L of hexanoic acid in a batch culture. However, high titer of hexanoic acid is toxic to the cellular growth. Therefore, the objective of this study is to improve hexanoic acid production of Megasphaera elsdenii by using reactive extraction method. The extraction of hexanoic acid was performed with 10 % (v/v) Alamine 336 in Oleyl alcohol as a solvent and this method led the selective removal of hexanoic acid from the fermentation broth with high recovery rate. Compared to conventional fermentation, in situ extractive fermentation showed a 5.7-folds higher production of hexanoic acid (25.83 g/L) due to immediate extraction of hexanoic acid. In addition, this extraction method was applied to fed-batch fermentation, resulting that the titer of hexanoic acid was increased up to about 70.04 g/L (15.5-folds higher). In conclusion, the efficient extraction process is crucial for fermentative production of hexanoic acid by Megasphaera elsdenii NCIMB 702410 using sucrose as a sole carbon.

Human adipose tissue-derived mesenchymal stem cells (hADSCs) have power to differentiate various cell types including chondrocytes, osteocytes, adipocytes, neuron, cardiomyocytes, and smooth muscle. In the present study, we characterize the functional expression of ion channels using TGF-β1-induced differentiation of hADSCs and provide the clues for differentiation to the vascular smooth muscle cells. The treatment of TGF-β1 to the hADSCs dramatically increased the expression levels of smooth muscle specific genes including α-smooth muscle actin (α-SMA), calponin, SM-MHC, smoothelin-B, myocardin, and h-caldesmon with contraction of collagen gel lattice. TGF- β1-induced differentiation of hADSCs appeared the Ca2+, big-conductance Ca2+- activated K+ (BKCa), and voltage-dependent K+ (Kv) currents comparing lack of these current in undifferentiated hADSCs. These appearing currents shared the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and western blotting revealed that L-type (Cav1.2) and T-type (Cav3.1, 3.2, 3.3) Ca2+ channels, which have known to express vascular SMCs, were dramatically increased in TGF-β1-induced differentiation of hADSCs with Cav β1 and Cav β3 subtypes. Although the expression changes in β-subtypes of BKCa channel were relatively various, the major α-subtypes of BKCa channel (KCa1.1) were increased in TGF-β 1-induced differentiation of hADSCs. The most Kv subtypes, which known to express in vascular SMCs, also dramatically increased in TGF-β1-induced differentiation of hADSCs. These results suggested that TGF-β1 induced differentiation of hADSCs into the contractile vascular SMCs with expression of vascular SMCs-like ion channels.

Introduction: Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hADSCs) to smooth muscle cells (SMCs). Materials and Methods: In the present study, we characterized contractile and ion channel properties of SMCs differentiated from hADSCs (hADSC-SMCs) as a result of SPC treatment, and we investigated the molecular mechanisms involved in the SPC-induced differentiation. Results and Discussion: Using in vitro collagen gel lattice contraction and whole cell patch clamp, we showed that the hADSC-SMCs expressed functional L-type voltage-gated Ca2+ channels and contractile activities in response to KCl, carbachol, and the L-type Ca2+ channel opener Bay K8644, whereas the L-type Ca2+ channel blocker nifedipine abrogated the contractility of hADSC-SMCs. Furthermore, hADSC-SMCs expressed functional big conductance Ca2+-activated K+ (BKCa) channels, and the BKCa channel blocker iberiotoxin potentiated the Bay K8644-stimulated contractility of the hADSC-SMCs, indicating that these cells exhibited SMC-like contractile characteristics. SPC activated RhoA in hADSCs and pretreatment with Rho kinase inhibitor Y27632 or by overexpression of dominant-negative mutants of RhoA or Rho kinase completely abtogated the SPC-induced differentiation of hADSCs into SMCs. SPC also increased the expression levels of myocardin-related transcription factor (MRTF)-A, a tanscription factor involved in smooth muscle differentiation, in hADSCs. Small interference RNA-mediated depletion of endogenous MRTF-A abolished the SPC-induced differentiation of hADSCs into SMCs. Furthermore, SPC promoted nuclear translocation of MRTF-A, and pharmacological inhibition of Tho kinase blocked this effect. These results suggest that SPC induced differentiation of hADSCs into contractile SMCs through a mechanism involving RhoA/Rho kinase-dependent nuclear translocation of MRTF-A.

In this study we tried to make a silk scaffold contained nano-HAp for dental tissue engineering. The silk scaffolds extracted sericin were coated with 0.3g, 0.15g, 0.03g of nano-HAp dissolved in PBS. Then, they were soaked in a 1% type I atelocollagen solution and air dried. They were crosslinked with 0.02% carbodiimide and lyophilized for 48 h. And they were sterilized by γ- irradiation at 10 KGy. DPSCs were seeded into silk scaffolds contained nano-HAp at a density of 2.8x104 cells/cm2 and cultured for 3weeks in growth medium. Then, they were cultured for 4weeks in differentiation medium and were transplanted in the nude mouse. The biopsy was processed at 8weeks. Col III and fibronectin, osteocalcin, osteopontin, osteonectin, osteoprotegerin and BMP-2 levels in the culture were greatest in the 0.15g of HAp. The calcification and the revelation of ostecalcin and osteopontin were better made in the silk scaffold contained 15mg of nano-HAp. We could make silk scaffold contained various concentration of nano-HAp. Among them, 15mg of nano-HAp was the most effective for osteogenesis. It will be a suitable substrate as biomaterial for bone tissue .

Introduction. The neuronal differentiation of bone marrow mesenchymal stem cells(BM-MSCs) has great potential for cellular therapies and might be useful in the treatment of diseases such as stroke or spinal cord injury. Many researches have investigated the effects of electromagnetic fields on neuronal differentiation of stem cells. Materials and Methods. Here, we analyzed the combined effects of ELFEMF(extremely low frequency electromagnetic fields) and differentiation medium (contatin hydrocortisone, insulin, forskolin, valproic acid, and KCl) on neural induction in human BM-MSCs. To induce neuronal differentication, BM-MSCs were incubated in differentiation medium for 1 day and treated for 7 days with 50 Hz, 100Hz, and 200 Hz electromagnetic fields subsequently. The cellular proliferation (MTT assay) investigated at different time points. Results. The results showed that exposure to neural differentiation medium and EMF decreased the cellular proliferation and enhanced the cellular differentiation. And we also examined immunocytochemistry, western blotting. We detected a significant increase of neuronal markers such as NF-L , Map2, and NeuroD1. Cells which were treated with 100 Hz EMF showed higher expression of NeuroD1, NF-L , and Map2 than 50 and 200 Hz. It confirmed by immunocytochemical analysis. And then, we confirmed that mean calcium current density increased after ELFEMF stimulation at 12 day of differentiation. We also found that ELFEMF increase the CREB phosphorylation at 24h of differentiation. Conclusion. Taken together, treatment of ELFEMF can be a good tool for neuronal differentiation and clinical implications.

Background : Visfatin (also known as pre-B-cell colony-enhancing factor, PBEF), is a novel adipokine, such as leptin, resistin, VEGF, IL-6, and FGF-2 that reported to play an important role in proangiogenic adipokine and neuroprotective effect. Methodology/Principal Findings : We investigated whether visfatin gene expression is associated with differentiation of neuronal cells. RT-PCR analysis, morphological observations, and immunostaining revealed that CoCl2, a hypoxic mimetic agent, at 75 microM increased the expression of visfatin gene and induced neurite outgrowth in PC12 rat pheochromocytoma cells. In addition, the treatement of visfatin stimulated neurite outgrowth with the increase in the levels of nerve growth factor (NGF) mRNA and protein. NGF upregulation by visfatin was prevented by an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Furthermore, visfatin-induced neurite outgrowth was also reduced by inhibition of ERK1/2 and NGF receptor TrkA pathways. Conclusions/Significance : Taken together, our results indicate that visfatin-induced neurite outgrowth is composed largely of two sequential steps: the induction of ERK1/2-dependent NGF gene expression by visfatin and the subsequent NGF-induced neuritogenesis. These data further suggest an integral role for visfatin-NGF signaling axis in modulating neurite outgrowth of neuronal cells.

Ligament-bone integration was important for ligament tissue reconstruction. Graft integration is required for successful reconstruction of the anterior cruciate ligament. We made a comparative study on the effect of silk scaffold and lyophilized collagen/HA substrate-containing silk scaffold (composite silk scaffold). As this result, proliferation, and bone-related marker expression in vitro were well observed in UC-MSC-seeded composite silk scaffolds compared to the cell-seeded silk scaffold. Further, silk and composite silk scaffolds were implanted into the knees of rabbits as artificial ligament, and they were harvested 24 weeks after implantation for assessing ligament-to-bone integration. It is thought that the lyophilized collagen/HA substrate is highly biocompatible in vitro and enhances wound healing in vivo.

Mesenchymal stem cells (MSCs) have been investigated as new cell- therapeutic solution due to their capacity that differentiate into cells of connective tissues. Many studies have proved that human bone marrow-derived MSCs (hBM-MSCs) have capacity of neural differentiate potential by treating mechanical stimulation. In our study, we chose electromagnetic fields as mechanical stimulus. Recently, magnetic iron oxide nano particles (MNPs) have been suggested new method for neural cell transplantation because MNP-labeled cells are non-invasive imaging for cell tracking and can delivery of therapeutic bio-molecules strongly. So we used PEG-phospholipid encapsulated magnetite (Fe3O4) nanoparticles on hBM- MSCs to improve their intracellular uptake and help the differentiate neural cells. So, first of all, we measured cell viability on hBM-MSCs treated with MNPs by MTT assay. Then, we exposed to 50Hz electromangetic fields in neurobasal media, DMEM/F12. After exposing, we analyzed by RT-PCR, western blotting using neural cell type-specific genes and antibodies. So these results suggest that electromagnetic fields enhances neural differentiation on hBM-MSCs incorporated with MNPs and it would be a effective method to differentiate neural cell.

Ligament-bone integration was important for ligament tissue reconstruction. Graft integration is required for successful reconstruction of the anterior cruciate ligament. We made a comparative study on the effect of silk scaffold and lyophilized collagen/HA substrate-containing silk scaffold (composite silk scaffold). As this result, proliferation, and bone-related marker expression in vitro were well observed in UC-MSC-seeded composite silk scaffolds compared to the cell-seeded silk scaffold. Further, silk and composite silk scaffolds were implanted into the knees of rabbits as artificial ligament, and they were harvested 24 weeks after implantation for assessing ligament-to-bone integration. It is thought that the lyophilized collagen/HA substrate is highly biocompatible in vitro and enhances wound healing in vivo.

Embryonic stem cells (ESCs) give rise to the possibility that stem cell therapy. However, before cell replacement therapies can be used, the problems with the tumorigenicity of ESCs must be solved. In the present study, we attempt to find biomarkers related with tumor formation of ESCs. We induced neuronal differentiation from H9, a hESCs, using a five-stage method and performed characterization of hESCs in each developing stages. We decided stage 3 as the step of cell sorting and performed separating cells on SSEA3 and CD133 using the MACS. In the results of in vivo assay with each sorted cells, CD133 positive and negative cells were not related with tumorigenesis. But groups injected the SSEA3 positive cells showed high levels of tumor forming rate as 100%, otherwise the group injected SSEA3 negative cells not detected tumorigenesis during periods of study. In these results, we recognized that SSEA3 related with undifferentiated status has important role in reduction of tumor formation on hESCs tumorigenesis, suggesting that these appears to be a useful biomarker for the assessment of the safety of stem cell-based therapy products using hESC.

Fermentation process could enhance biological activities, specifically antioxidant and antimicrobial properties by modifying naturally occurring constituents of isoflavons, saponins, phytosterols, and phenols. TLC and HPLC showed the increase of rutin and quercetin, quercitrin due to the fermentation of Houttuynia cordata. This study was aimed to investigate the effect of fermented Houttuynia cordata (FH) on the improvement of lipid metabolism in rats fed high fat diet. The levels of body weight, AST, ALT, total cholesterol, HDL-cholesterol, LDL cholesterol, triglyceride(TG) and atherogenic index(AI) were measured in serum. Malondialdehyde(MDA) was measured in liver homogenate. High fat diet fed rat markedly increased the levels of AST, ALT, total cholesterol, LDL cholesterol, TG, AI and MDA, and significantly decreased the levels of HDL-C. But FH and NFH(non-fermented Houttuynia cordata) pretreatment decreased levels of AST, ALT, total cholesterol, LDL cholesterol, TG, AI and MDA respectively and increased those of HDL-C. FH group showed the most ameliorated state of steatosis making the decrease of small lipid droplets and the improvement of vacuolar degeneration in liver tissue. These results showed the FH had the effects of anti-obesity in obese rat model.

As an alternative treatment of Diabetes Melitus, xeno-transplantation of insulin-secreting pancreatic islets has been emerged. Microencapsulation of islets is essential in order to prevent islets from attack of host immune system. However, large amount of empty capsule after microencapsulation process using alginate as a biocompatible material is produced under air-driven system, which causes enlargement of transplantation volume. In this research, empty alginate capsules and encapsulated islets were separated of viability using density-gradient solution. Also, its function and viability were confirmed to check toxicity of density-gradient solution and harmness of separation process.