Shin Sik Choi

Enhancement of recombinant human EPO production and glycosylation in serum-free suspension culture of CHO cells through expression and supplementation of 30Kc19

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14xa0%, respectively), while the control cells produced predominantly bi-antennary branching (70xa0%). About 53xa0% of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77xa0%, whereas only 61xa0% of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6xa0% after the addition of 0.2xa0mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins.

A protein delivery system using 30Kc19 cell-penetrating protein originating from silkworm

Cell-penetrating protein and its protein transduction domain have been used to deliver drugs and proteins into the cells via receptor-independent endocytosis. A number of cell-penetrating proteins including TAT derived from HIV-1 virus, VP22 from herpes simplex virus and Antennapedia from drosophila have been discovered. Here, we report a cell-penetrating protein, 30Kc19, originating from the hemolymph of silkworm, Bombyx mori. The 30Kc19 is the first cell-penetrating protein that has been found in insect hemolymph. When the 30Kc19 protein produced from recombinant Escherichia coli was supplemented into the medium for mammalian cell culture, 30Kc19 efficiently penetrated into various types of cells and localized at subcellular compartments including mitochondria and cytoplasm. 30Kc19 also delivered cargo proteins such as green fluorescence protein into the cells even though cargo proteins are not able to penetrate into cells by themselves. In addition to the inxa0vitro system, 30Kc19 exhibited the protein transduction property inxa0vivo. When 30Kc19 was intraperitoneally injected into mice, 30Kc19 delivered cargo proteins into various organ tissues of model animals without producing toxicity. Therefore, 30Kc19 has a great potential as a cell-penetrating protein that can be used as a medicinal tool to deliver cargo molecules including proteins into the target organ tissues in the body.

Antioxidant effect of protein-free silkworm hemolymph extract in mitochondrial membrane potential

Silkworm was used in the sericulture industry and also processed for food sources in some of Asian countries. In previous studies, it was reported that the 30K protein in silkworm, Bombyx mori hemolymph inhibited animal cell apoptosis. In this work, the antioxidative function of silkworm hemolymph was investigated using its protein-free extract. H2O2-induced ROS and O2·− were efficiently scavenged in Chinese hamster ovary cells when the culture medium was supplemented with silkworm hemolymph. The hemolymph extract prepared by deproteinization through ethanol precipitation completely scavenged DPPH radicals (IC50=0.025 mg/mL). When the extract was supplemented with protein-free medium, oxidative damage to mitochondria was efficiently diminished with the maintenance of the mitochondrial membrane potential. Lutein was identified as the major antioxidative constituents in the extract (52.06 μg/mg).

The cultivation of Anabaena variabilis in a bubble column operating under bubbly and slug flows.

In a bubble column reactor with an inner diameter of 6cm and a height of 63cm for the culture of cyanobacteria two different shapes of bubbles can be generated, resulting in bubbly flow or slug flow. Growth of Anabaena variabilis under slug flow (1.9g/l/day) was 1.73 times higher than that under bubbly flow (1.1g/l/day) when the specific irradiation rate was maintained above 10μmol/s/g dry cell. Although a stepwise increase in superficial gas velocity enhanced the average cell growth rate under bubbly flow by 1.57 times, the average cell growth rate during the deceleration phase under bubbly flow (1.98g/l/day) was 0.61 times smaller than that under slug flow (3.22g/l/day). These results demonstrate that the bubble shape in the slug flow was advantageous in regards to the radial circulation of cells.

C. elegans -on-a-chip for in situ and in vivo Ag nanoparticles’ uptake and toxicity assay

Nanomaterials are extensively used in consumer products and medical applications, but little is known about their environmental and biological toxicities. Moreover, the toxicity analysis requires sophisticated instruments and labor-intensive experiments. Here we report a microfluidic chip incorporated with the nematode Caenorhabditis elegans that rapidly displays the changes in body growth and gene expression specifically responsive to the silver nanoparticles (AgNPs). C. elegans were cultured in microfluidic chambers in the presence or absence of AgNPs and were consequently transferred to wedge-shaped channels, which immobilized the animals, allowing the evaluation of parameters such as length, moving distance, and fluorescence from the reporter gene. The AgNPs reduced the length of C. elegans body, which was easily identified in the channel of chip. In addition, the decrease of body width enabled the worm to advance the longer distance compared to the animal without nanoparticles in a wedge-shaped channel. The transgenic marker DNA, mtl-2::gfp was highly expressed upon the uptake of AgNPs, resulting in green fluorescence emission. The comparative investigation using gold nanoparticles and heavy-metal ions indicated that these parameters are specific to AgNPs. These results demonstrate that C. elegans-on-a-chip has a great potential as a rapid and specific nanoparticle detection or nanotoxicity assessment system.

Enhanced biological activity of carotenoids stabilized by phenyl groups

Carotenoids are lipid soluble food ingredients with multifunction including antioxidant and anticancer activities. However, carotenoids are destructively oxidized upon reaction with radicals resulting in toxic effects on biological systems. Two synthetic carotenoids (BAS and BTS) containing the aromatic phenyl groups with a para-substituent (OMe and Me, respectively) at C-13 and C-13 position were prepared in order to overcome a structural instability of carotenoid. Both BAS and BTS exerted stronger radical scavenging activity than β-carotene in DPPH and ABTS assays. In particular, BTS significantly reduced in vivo ROS (reactive oxygen species) levels and improved body growth and reproduction of Caenorhabditiselegans. BTS has a great potential for the advanced and modified carotenoid material with stability leading to enhanced bioavailability.

Protective effects of silkworm hemolymph extract and its fractions on UV-induced photoaging

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.

A triangle study of human, instrument and bioelectronic nose for non-destructive sensing of seafood freshness

Because the freshness of seafood determines its consumer preference and food safety, the rapid monitoring of seafood deterioration is considered essential. However, the conventional analysis of seafood deterioration using chromatography instruments and bacterial colony counting depends on time-consuming and food-destructive treatments. In this study, we demonstrate a non-destructive and rapid food freshness monitoring system by a triangular study of sensory evaluation, gas chromatography-mass spectroscopy (GC-MS), and a bioelectronic nose. The sensory evaluation indicated that the acceptability and flavor deteriorated gradually during post-harvest storage (4u2009°C) for 6 days. The GC-MS analysis recognized the reduction of freshness by detecting a generation of dimethyl sulfide (DMS) from the headspace of oyster in a refrigerator (4u2009°C) at 4 days post-harvest. However, the bioelectronic nose incorporating human olfactory receptor peptides with the carbon nanotube field-effect transistor sensed trimethylamine (TMA) from the oyster at 2 days post-harvest with suggesting early recognition of oysters’ quality and freshness deterioration. Given that the bacterial species producing DMS or TMA along with toxins were found in the oyster, the bacterial contamination-driven food deterioration is rapidly monitored using the bioelectronic nose with a targeted non-destructive freshness marker.

Antifungal activity of nano and micro charcoal particle polymers against Paecilomyces variotii, Trichoderma virens and Chaetomium globosum

This study investigates the antifungal activity of a polymer integrated with nano-porous charcoal particles against Paecilomyces variotii, Chaetomium globosum, Trichoderma virens, which are all filamentous fungi. The charcoal polymers were prepared by combining charcoal powders with plastic resin under a vacuum to form charcoal particle protrusions on the polymer surface. The mycelial growth of P. variotii and T. virens exhibited a reduction of 10 and 30%, respectively, after the conidia were pre-treated with charcoal polymers, and in particular, no mycelial growth was found in C. globosum during 5 days of culture. The adsorption of Ca(2+) into charcoal was suggested to inhibit growth due to the reduction in the flux of calcium ions (Ca(2+)) into the hyphae. In 5 h, about 15 mM of Ca(2+) were removed from CaCl2 solution with 0.2 g/mL of polymers, and the nano-sized pores of the charcoals on the polymer were responsible for the Ca(2+) adsorption.

Effect of lactic acid bacteria on intestinal E. coli in Caenorhabditis elegans

Lactic acid bacteria (LAB) prevent host organism from digestion-related illnesses, such as constipation, diarrhea, irritable bowel syndrome, and enteric infection. In this study, we investigated whether dietary LAB culture product reduced the level of viable intestinal E. coli, using nematode Caenorhabditis elegans as host animal. Among four LAB strains, Lactobacillus plantarum product most significantly reduced the number of E. coli living in the intestine of both 2-day and 7-day old adult C. elegans. The lower number of E. coli in the digestion system by LAB products consequently resulted in decrease of living E. coli in the excrement. LAB products led to an improvement of body growth, survival ratio and reproduction in C. elegans. Based on in vitro and in vivo antioxidant activity analysis, it was also suggested that LAB products reduce oxidative stress in the body.