Enhanced lipase-catalyzed synthesis of sugar fatty acid esters using supersaturated sugar solution in ionic liquids.

A solvent-mediated method (SMM) was used to prepare supersaturated sugar solutions in hydrophobic and mixture of hydrophilic/hydrophobic ionic liquids (ILs), namely, [Bmim][Tf2N] and [Bmim][TfO]/[Bmim][Tf2N], respectively. In this method, sugars were first solubilized in a mixture of organic solvent and water (i.e. methanol:water, 1:1 v/v), and then added to [Bmim][Tf2N] and/or [Bmim][TfO]/[Bmim][Tf2N] mixture. Supersaturated sugar solution in ILs were obtained by removing organic solvents and water under vacuum evaporation. Sugar solubilities in ILs, especially in hydrophobic IL ([Bmim][Tf2N]) and in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM were greater than in ILs prepared using water-mediated method (WMM), which suggested methanol aided sugar solvation in hydrophobic media. In addition, interactions between glucose molecules and between glucose and methanol, water, and IL were investigated by all-atom molecular dynamics (MD) simulation. The MD simulation results showed that initial water and water/methanol molecules around glucose were gradually replaced by IL anions. Notably, SMM resulted in stronger interaction between IL anions and glucose than WMM, which was attributed to greater solubility of sugar in ILs prepared by SMM. Resultantly, the productivity of lipase-catalyzed production of glucose laurate using supersaturated glucose solution in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM was at least 1.76-fold greater than that obtained in IL mixture prepared by WMM.

Experimental Tracheal Replacement Using 3-dimensional Bioprinted Artificial Trachea with Autologous Epithelial Cells and Chondrocytes.

Various treatment methods for tracheal defects have been attempted, such as artificial implants, allografts, autogenous grafts, and tissue engineering; however, no perfect method has been established. We attempted to create an effective artificial trachea via a tissue engineering method using 3D bio-printing. A multi-layered scaffold was fabricated using a 3D printer. Polycaprolactone (PCL) and hydrogel were used with nasal epithelial and auricular cartilage cells in the printing process. An artificial trachea was transplanted into 15 rabbits and a PCL scaffold without the addition of cells was transplanted into 6 rabbits (controls). All animals were followed up with radiography, CT, and endoscopy at 3, 6, and 12 months. In the control group, 3 out of 6 rabbits died from respiratory symptoms. Surviving rabbits in control group had narrowed tracheas due to the formation of granulation tissue and absence of epithelium regeneration. In the experimental group, 13 of 15 animals survived, and the histologic examination confirmed the regeneration of epithelial cells. Neonatal cartilage was also confirmed at 6 and 12 months. Our artificial trachea was effective in the regeneration of respiratory epithelium, but not in cartilage regeneration. Additional studies are needed to promote cartilage regeneration and improve implant stability.

3D Bioprinted Artificial Trachea with Epithelial Cells and Chondrogenic-Differentiated Bone Marrow-Derived Mesenchymal Stem Cells.

Tracheal resection has limited applicability. Although various tracheal replacement strategies were performed using artificial prosthesis, synthetic stents and tissue transplantation, the best method in tracheal reconstruction remains to be identified. Recent advances in tissue engineering enabled 3D bioprinting using various biocompatible materials including living cells, thereby making the product clinically applicable. Moreover, clinical interest in mesenchymal stem cell has dramatically increased. Here, rabbit bone marrow-derived mesenchymal stem cells (bMSC) and rabbit respiratory epithelial cells were cultured. The chondrogenic differentiation level of bMSC cultured in regular media (MSC) and that in chondrogenic media (d-MSC) were compared. Dual cell-containing artificial trachea were manufactured using a 3D bioprinting method with epithelial cells and undifferentiated bMSC (MSC group, n = 6) or with epithelial cells and chondrogenic-differentiated bMSC (d-MSC group, n = 6). d-MSC showed a relatively higher level of glycosaminoglycan (GAG) accumulation and chondrogenic marker gene expression than MSC in vitro. Neo-epithelialization and neo-vascularization were observed in all groups in vivo but neo-cartilage formation was only noted in d-MSC. The epithelial cells in the 3D bioprinted artificial trachea were effective in respiratory epithelium regeneration. Chondrogenic-differentiated bMSC had more neo-cartilage formation potential in a short period. Nevertheless, the cartilage formation was observed only in a localized area.

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids.

The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.

Ionic liquids as novel solvents for the synthesis of sugar fatty acid ester.

Sugar fatty acid esters are bio-surfactants known for their non-toxic, non-ionic, and high biodegradability . With great emulsifying and conditioning effects, sugar fatty acids are widely used in the food, pharmaceutical, and cosmetic industries. Biosynthesis of sugar fatty acid esters has attracted growing attention in recent decades. In this study, the enzymatic synthesis of sugar fatty acid esters in ionic liquids was developed, optimized, and scaled up. Reaction parameters affecting the conversion yield of lipase-catalyzed synthesis of glucose laurate from glucose and vinyl laurate (i.e. temperature, vinyl laurate/glucose molar ratio, and enzyme loads) were optimized by response surface methodology (RSM). In addition, production was scaled up to 2.5 L, and recycling of enzyme and ionic liquids was investigated. The results showed that under optimal reaction conditions (66.86 °C, vinyl laurate/glucose molar ratio of 7.63, enzyme load of 73.33 g/L), an experimental conversion yield of 96.4% was obtained which is close to the optimal value predicted by RSM (97.16%). A similar conversion yield was maintained when the reaction was carried out at 2.5 L. Moreover, the enzymes and ionic liquids could be recycled and reused effectively for up to 10 cycles. The results indicate the feasibility of ionic liquids as novel solvents for the biosynthesis of sugar fatty acid esters.

Refolding of laccase in dilution additive mode with copper-based ionic liquid.

Ionic liquids (ILs) are molten salts which do not crystallize at room temperature. Tunable physicochemical properties of ILs including hydrophobicity and polarity facilitate their applications in many biological processes. In this study, a copper-based IL was employed in order to enhance the refolding efficiency of laccase from Trametes versicolor which requires copper as a cofactor. When 1-ethyl-3-methylimidazolium trichlorocuprate ([EMIM][CuCl3]) was added to refolding buffer instead of urea, the laccase refolding yield was improved more than 2.7 times compared to the conventional refolding buffer which contains urea. When the refolding of laccase was carried out at different temperatures (4, 25, and 37 °C), the highest refolding yield was obtained at 25 °C. At low temperature, two conflicting effects, i.e., suppression of the aggregate formation and decrease of folding rate, influence the protein refolding. In contrast, a copper-based IL did not enhance the refolding of lysozyme, a non-copper-containing protein. From these results, we can conclude that this copper-based IL, [EMIM][CuCl3], was exclusively effective on the refolding process of a copper-containing protein.

Virus-like particle vaccine protects against H3N2 canine influenza virus in dog.

In the present study, virus-like particles (VLPs) were evaluated as a candidate veterinary vaccine against canine influenza virus (CIV) subtype H3N2. Specific pathogen-free (SPF) beagle dogs received a single injection of a VLP vaccine containing hemagglutinin (HA) and M1 protein of CIV H3N2 (H3 HA VLP). The vaccine was tested at 3 different doses with an adjuvant and 1 dose without an adjuvant. To evaluate the immunogenicity and protective efficacy of the H3 HA VLP vaccine, we performed hemagglutination inhibition tests to determine serological immune responses and conducted challenge studies using SPF beagle dogs. The addition of Montanide ISA 25 adjuvant significantly increased the immunogenicity of the H3 HA VLP vaccine. The experimental infection study showed that a single dose of H3 HA VLP vaccine induced protection against wild-type virus challenge in dogs. These results provide support for continued development of the VLP as an animal vaccine against influenza virus.

miR-196a regulates proliferation and osteogenic differentiation in mesenchymal stem cells derived from human adipose tissue.

The elucidation of the molecular mechanisms that govern the differentiation and proliferation of human adipose tissue-derived mesenchymal stem cells (hASCs) could improve hASC-based cell therapy. In this study, we examined the roles of microRNA (miRNA)-196a on hASC proliferation and osteogenic differentiation. Lentiviral overexpression of miR-196a decreased hASC proliferation and enhanced osteogenic differentiation, without affecting adipogenic differentiation. Overexpression of miR-196a decreased the protein and mRNA levels of HOXC8, a predicted target of miR-196a. HOXC8 expression was decreased during osteogenic differentiation of hASCs, and this decrease in HOXC8 expression was concomitant with an increase in the level of miR-196a. In contrast, inhibition of miR-196a with 2'-O-methyl-antisense RNA increased the protein levels of HOXC8 in treated hASCs and was accompanied by increased proliferation and decreased osteogenic differentiation. The activity of a luciferase construct containing the miR-196a target site from the HOXC8 3'UTR was lower in LV-miR196a-infected hASCs than in LV-miLacZ-infected cells. RNA interference-mediated downregulation of HOXC8 in hASCs increased their proliferation and decreased their differentiation into osteogenic cells, without affecting their adipogenic differentiation. Our data indicate that miR-196a plays a role in hASC osteogenic differentiation and proliferation, which may be mediated through its predicted target, HOXC8. This study provides us with a better knowledge of the molecular mechanisms that govern hASC differentiation and proliferation.

COMP-Ang1, angiopoietin-1 variant protects radiation-induced bone marrow damage in C57BL/6 mice.

Angiopoietin-1 (Ang1) is a vasculogenic factor which is signaled through the endothelial and bone marrow cell-specific, Tie2 receptor tyrosine kinase and has potential therapeutic applications for the induction of angiogenesis, enhancing endothelial cell survival, and preventing vascular leakage. In this study, we examined whether Ang1 directly exhibits bone marrow protection after ionizing radiation (IR) using an adenoviral vector of COMP-Ang1 (Ad-COMP-Ang1). This is a variant of Ang1 by replacement of the N-terminal portion of Ang1 with short coiled-coil domains of cartilage oligomeric matrix protein-Angiopoietin 1 (COMP-Ang1) which are, long enough for oligomerization but short enough to avoid problems of aggregation and insolubility. A spleen colony assay after 4.5 Gy whole body radiation, indicated that COMP-Ang1 significantly increased the mean colony numbers. Both the decrease in bone marrow cellularity and increased TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) positive cells produced by radiation in bone marrow were significantly inhibited by COMP-Ang1 transfer. The expression of the ligands of Ang1 and Tie2 receptors were increased by radiation and, the COMP-Ang1 transfer potentiated this protein expression. Pre-treatment of Ang1 could be beneficial in protecting bone marrow from damage by radiation and COMP-Ang1 may be an effective alternative to native Ang1 for therapeutic purposes.