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Previous studies have shown that functionalized gold nanoparticles (AuNPs) can be used as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNA to living systems. Here, we report the ability of functionalized AuNP to deliver RNA aptamers into the nuclei of human cells. An in vitro-synthesized RNA aptamer specific to the β-catenin protein was delivered into the HepG2 human cell line more efficiently via functionalized AuNP than liposome-based delivery, and resulted in nearly complete inhibition of β-catenin binding to the p50 subunit of NF-κB in the nucleus. This inhibition led to repression of NF-κB p50-dependent transcription of CRP. Also, the β-catenin aptamer in the nucleus led to down-regulation of β-catenin-mediated transcriptional activity through the TCF complex and resulted in decrease in the levels of cyclin D, and c-myc mRNA by ∼47% and ∼57%, respectively. In addition, we used functionalized AuNP to deliver another RNA aptamer targeted to the p50 subunit of NF-κB into the A549 human cell line, and this was sufficient to induce apoptosis of the cells. Our findings demonstrate that AuNP GDS can be used to deliver small, highly structured RNA aptamers into the nucleus of human cells where they modulate the activity of transactivators by interacting with target proteins.

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.

Several molecular techniques have been used to study viruses under different environmental conditions and to examine the genetic diversity of natural virus communities. Here, natural marine virus samples were collected from six different southern coastal regions of Korea and subjected to PCR amplification with five different degenerate primers specific for either the DNA polymerase or capsid protein gene of algal viruses. PCR products ranging from 300 to 700 bp were observed on agarose gel analysis, and major PCR bands were purified and cloned. PCR using primers specific for the viral DNA polymerase gene and for the coat protein gene yielded 332 and 366 clones, respectively. Of the clones analyzed, 147 (44%) DNA polymerase gene clones and 326 (89%) coat protein clones showed similarity to known virus genes. Clustering and assembly revealed 23 and 38 unique sequences for the DNA polymerase and coat protein genes, respectively. BLASTX and phylogenetic analyses of these sequences revealed close relationships with various virus groups, including one of the major algal virus groups, Phycodnaviridae . Pairwise nucleotide comparisons among sequences classified into the same group revealed the extent of genetic diversity in both polymerase and coat protein gene sequences. These results indicated the presence of genetic diversity within similar virus groups in the marine system. Additionally, PCR products with the same sequences were recovered from different locations, indicating the presence of the same virus in different geographic locations in the southern coastal region of the Korean Peninsula.

Bio-indigo was produced in 3000-L batch and 5-L continuous fermentation of a recombinant Escherichia coli DH5α harboring a novel gene encoding flavin-containing monooxygenase (FMO). Batch fermentation in a 3000-L fermenter produced 911 ± 22 mg L−1 of indigo from 2 g L−1 tryptophan as a substrate (yield 46.9%) under the following culture conditions as follows: culture temperature 30 °C, pH 7.0, agitation speed 200 rpm, and aeration 3 vvm. Sufficient oxygen (aeration rate and agitation speed) was critical for bio-indigo production. For continuous fermentation in a 5-L fermenter, the volumetric productivity was found to be 11.3 mg L−1 h−1 up to 110 h (final accumulated bio-indigo was 23 g) with a constant dilution rate (D) of 0.084 h−1 (constant feeding rate of 0.167 L h−1 with medium containing 3 g L−1 tryptophan). Recombinant E. coli cells have the ability to withstand the toxicity of high concentration of accumulated indigo in batch fermentation. In continuous fermentation, the recombinant cells exhibited high plasmid stability up to 110 h, after which they lost the plasmid.

The objective of this study was to develop chromatographic methods for the determination of the modification degree and the characterization of poly(ethylene glycol)-modified polyamidoamine dendrimers (PEG–PAMAMs). The PEG–PAMAMs were prepared by reacting PAMAM generation 4 with monomethoxy PEG–nitrophenyl carbonate (mPEG–NPC). The modification degrees of PEG–PAMAMs were determined by quantifying 4-nitrophenol released from mPEG–NPC after PEGylation reaction using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The PEG–PAMAMs, which have poor UV absorbances, were characterized by HPLC with charged aerosol detection. This study demonstrates that the combination of these two detectors is a powerful tool for the preparation and characterization of PEG–PAMAMs.

Response surface methodology (RSM) based on the 23 factorial central composite design (CCD) was applied to optimize the conditions of enzymatic saccharification and ethanol fermentation using food waste. Optimum conditions were found to be saccharification pH of 5.20, enzyme reaction temperature of 46.3 °C, enzyme concentration of 0.16% (v/v), fermentation pH of 6.85, fermentation temperature of 35.3 °C, and fermentation time of 14 h. The model predicted that maximum concentration of reducing sugar and ethanol under the above optimum conditions were 117.0 g reducing sugar/L and 57.6 g EtOH/L, respectively. Experimental results were in close agreement with model prediction with 120.1 g reducing sugar/L and 57.5 g EtOH/L, respectively.

In this study, a volumetric scale-up of this system was designed and built on a field pilot-scale (total digester volume 10 m(3)), with the results from the field pilot-scale experiments compared with those from the bench-scale (total digester volume 0.4 m(3)) process prior to scale-up. The reduction rate of total chemical oxygen demand (tCOD) and the maximum methane content produced in the biogas from the bench-scale system were 90.6% and 72%; whereas those from the field pilot-scale system were 90.1% and 68%, respectively. The estimated methane yields were 282 and 254 l CH(4)/kg tCOD(degraded) in bench and field pilot-scale fermentation systems, respectively. These results indicate that the three stage fermentation system developed in this study can be applied as a commercial process for the disposal of food waste in view of process stability.

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP T , and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli . To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l −1 of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2 n central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l −1 , yeast extract 4.5 g l −1 and sodium chloride 11.4 g l −1 . In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.

Indirubin, an active ingredient of a traditional Chinese recipe Danggui Longhui Wan, has been known as a CDK inhibitor competing with ATP for binding to the catalytic site of cyclin-dependent kinases (CDKs). Since CDKs, a group of serine/threonine kinases forming active heterodimeric complexes with cyclins, are key regulators of the cell cycle regulation, therapeutic interventions targeting CDKs have been stimulated for the treatment of proliferative diseases, such as cancer, psoriasis, and for the prevention of chemotherapy-associated side effects, such as alopecia. A series of novel indirubin analogs was synthesized and evaluated for anti-proliferative and CDK2 inhibitory activities. Among the indirubin derivatives tested in the growth inhibitions against several human cancer cell lines, 5-nitro, halide, and bulky group containing acylamino substituted analogs showed high anti-proliferative effects. Selected analogs showing potent anti-proliferative activities were evaluated further in the CDK2 enzyme assay, which resulted in the discovery of potent CDK2 inhibitors.

H 2 S removal by immobilized cell bioreactor system was investigated using continuous glass column bioreactor with diameter of 90 mm and with a height of 1000 mm. Thiobacillus sp. IW was encapsulated inside the Ca-alginate bead in a continuous H 2 S removal bioreactor system and an optimum bioreactor performance was achieved at 1.5 l media volume, under the following conditions: operating temperature of 30 °C, pH 8, bead volume of 30%, and bead diameter of 2 mm. The most active cell-immobilized bead was made in a continuous bead maker at 3% Na-alginate, 3% CaCl 2 , and 2.0 g/l of cell concentration. We could remove H 2 S in the inlet stream completely up to 600 ppm of influent concentration. It could be confirmed by scanning electron microscope (SEM) that not only did the cells grow well inside the pores of the bead but they also grew out to the surface of the bead after the H 2 S removal reaction. It has so far successfully run the continuous H 2 S removal bioreactor for up to 240 h without pH control.

It has been suggested that blood vessel formation is an important event coupled to bone formation. The expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been shown to be greatly stimulated in osteoblasts by hypoxic stimuli such as deprivation of oxygen and treatment with cobalt. In other cell types, hypoxia-inducible factor-1 (HIF-1) that binds hypoxia-response element (HRE) has been shown to mediate gene expression induced by hypoxic stimuli. In this study, we investigated the effects of hypoxic stimuli on HIF-1, HRE, and VEGF in osteoblastic cell lines. Exposure of these cells to hypoxia or cobalt resulted in a great increase in the protein level of HIF-1alpha and the gene expression of VEGF. Transforming growth factor-beta1, prostaglandin E2, dexamethasone, and 1,25-dihydroxyvitamin D3 that have been shown to regulate VEGF gene expression in osteoblasts had no effect on HIF-1alpha induction. Blocking the enzymatic activity of phosphatidylinositol 3-kinase, p38, MEK-1 did not have any effect on the cobalt-stimulated increase of HIF-1alpha in these cells. In contrast, N-acetylcysteine (NAC), a scavenger of reactive oxygen species, abolished the cobalt induction of HIF-1alpha and that of the VEGF and a HRE-driven reporter genes. However, the hypoxia responses were not affected by NAC. These findings suggest that hypoxia and cobalt can induce VEGF gene expression in osteoblasts by increasing the level of HIF-1alpha protein through different mechanisms.