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In this study, we investigated the effect of sample pretreatments (ultrasonication and alkaline extraction) on total organic carbon (TOC) measurements for water samples containing suspended solids (SS) of four different origins (algae, soil, sewage sludge, and leaf litter) to more clearly assess the impact of particulate organic carbon (POC) in water. The effects each of ultrasonication (power, pulse, etc.) and alkaline extraction condition (concentration, time, etc.) on the TOC recovery and precision were investigated, and the results were compared with those of a new sample pretreatment method combining both methods. Alkaline treatment (0.01 mol/L NaOH) showed higher precision than ultrasonication (100/5 on/off pulse), and notably, the differences among the measured TOC values in samples of different origins were also further reduced in the alkaline treatment. This suggests that the ultrasonic pretreatment results can be mainly attributed to the increase in POC recovery through particle size reduction, whereas the alkaline treatment results are achieved through the enhancement of POC solubilization. It is also particularly noteworthy that a higher TOC recovery of 87.6% ± 7.4% with a higher precision of 8.4% could be obtained using the combined method, compared to each treatment (ultrasonic: TOC recovery 34.7%, relative standard deviation 63.1%; alkaline: 49.6% and 23.0%, respectively). Thus, simultaneous pretreatment with ultrasonication and alkaline extraction is expected to increase the oxidation rate of organic matter and the homogeneity of the samples, minimizing the loss of POC measurement values, and thereby improving the reliability of the TOC measurements of water samples containing SS.

Particulate organic matter (POM) in water systems can be converted into dissolved organic matter (DOM) through various pathways depending on its properties and transformation. Thus, information on the behavior of POM is crucial for fully understanding water systems and the carbon cycle. In this study, the effects of particle size and the source of POM, as well as photochemical and microbial changes in DOM characteristics subsequently released from POM were evaluated using various spectral indices, excitation-emission matrix combined with parallel factor analysis components, and principal component analysis. The amount of dissolved organic carbon (DOC) released from POM during suspension was significantly associated with the carbon content of POM (p0.05). The amount of DOC (mg-C/g-SS) decreased in mineral-bound POM as a result of microbial degradation but increased in biogenic POM as a result of microbial dissolution, owing to the structural differences in organic matter from different sources. Mineral-bound POM showed more DOC production by photochemical desorption than microbial degradation, whereas biogenic POM displayed the opposite trend. The DOM derived from fine POM had more humified terrestrial humic-like substances than those derived from coarse POM. Principal components 1 and 2 were associated with DOC production and degree of humification, respectively. The increase in the degree of aromaticity and humification of organic matter was higher in mineral-bound POM by photochemical desorption of highly humified organic matter and in the biogenic POM by microbial dissolution. In conclusion, this study was able to provide basic information on the transformation of POM, thus, it is expected to broaden the knowledge of the biogeochemical cycle of organic matter.

Various biochars (BCs) have been developed to remove heavy metals contained in road runoff; however, there is insufficient information regarding the competitive adsorption efficiency of modified BC with regard to heavy metals due to a lack of comparative evaluation based on BC properties and modification methods. In this study, three different types of BC (RBC: rice husk, WBC: wood chip, MBC: mixture) were modified following five different methods: acidic, alkaline, oxidic, and manganese oxide (MnOx) and iron oxide (FeOx) impregnation. The changes in the physicochemical and morphological properties of the modified BC were investigated, and the adsorption characteristics of three heavy metals (Cd, Pb, and Zn) under single and mixed conditions were compared and evaluated. The improvements in the BC properties varied for different BC types and modification methods; in particular, alkaline and manganese modification caused substantial the changes in the surface area and functional groups (such as aromatic ring, –OH, and Mn–O groups). The BC prepared by manganese oxide impregnation absorbed a high amount of heavy metals (>9.15 mg/g) even under mixed conditions through cation exchange and surface complexation. The distribution coefficient (Kd) of heavy metals was high in the order of Pb > Cd > Zn; thus, the adsorption of Pb replaced that of Zn in competitive adsorption due to the difference in their affinity to BC. Therefore, the results suggest that BC prepared by manganese oxide impregnation is suitable for removing heavy metals from road runoff, as it maintained high heavy metals adsorption regardless of the BC material, even under competitive conditions.

Particulate matter (PM) has long-term effects on water quality compared to dissolved matter (DM) during downstream transfer after inflows into an aquatic environment. In the present study, the characteristics, behavior, and effects of PM from an urban watershed under photo-irradiation were investigated through sequential resuspensions before being compared. Changes in the organic matter content, heavy metals (Mn, Fe, Zn, Pb), spectroscopic indices (SUVA254, slope ratio (SR), humidification index (HIX), fluorescence index (FI), and biological index (BIX)), excitation-emission matrix combined with parallel factor analysis components (EEM-PARAFAC), and disinfection by-product formation potential (DBPFP) were analyzed. According to our results, light enhanced the release of organic matter from PM but reduced dissolved heavy metals. The PMU affected by urban-derived pollutants (i.e., rainfall particles, road-deposited sediment, sewer-pipeline-deposited sediment) exhibited higher quantities of terrestrial humic-like organic matter than PMR, which contains base particles from riverines (i.e., soil, sediments). For the PMU, the humic-like fluorescent components (C1 and C2) enhanced under light conditions with every resuspension, whereas the components decreased in the PMR. Consistent with the PARAFAC results, the trihalomethane formation potential (THMFP) of the PMU was enhanced by approximately 2.8 times more than that of the PMR, and exhibited a high correlation with the fluorescent components (C1, r = 0.81, p

Core–shell structured magnetic nanoparticles combine hard and soft phases to improve energy efficiency. The mutual interaction of the two phases can lead to the exchange spring effect, leading to higher magnetic energy. In this regard, synthesis of Nd2Fe14B-based core–shell-structured powders have proven to be elusive, due to the relatively reactive nature of this phase. In this study, a process has been established for successfully coating the surface of Nd2Fe14B powders with a FeCo layer using the galvanic displacement method. Initially, a binary phase magnetic powder was synthesized containing Nd2Fe14B and Nd2Fe17 phase. Subsequently, the powders were coated using a Co precursor at 303 K. During coating, the metastable Nd2Fe17 phase was dissolved, and the Fe ions were released into the solution. Subsequently, the Fe ions deposited together with the Co ions on the surface of Nd2Fe14B powder to form a FeCo shell. The deposited layer thickness and composition was confirmed using TEM analysis.

This study investigated the birnessite (&delta, MnO2) catalyzed oxidative removal of 1,4-naphthoquinone (1,4-NPQ) in the presence of phenolic mediators, specifically, the kinetics of 1,4-NPQ removal under various conditions was examined, and the reaction pathway of 1,4-NPQ was verified by liquid chromatography&ndash, tandem mass spectrometry (LC&ndash, MS/MS). The removal rate of 1,4-NPQ by birnessite-catalyzed oxidation (pH = 5) was faster in the presence of phenolic mediators with electron-donating substituents (pseudo-first-order initial stage rate constant (k1) = 0.380&ndash, 0.733 h&ndash, 1) than with electron-withdrawing substituents (k1 = 0.071&ndash, 0.244 h&minus, 1), and the effect on the substituents showed a positive correlation with the Hammett constant (&Sigma, &sigma, ) (r2 = 0.85, p &lt, 0.001). The rate constants obtained using variable birnessite loadings (0.1&ndash, 1.0 g L&minus, 1), catechol concentrations (0.1&ndash, 1.0 mM), and reaction sequences indicate that phenolic mediators are the major limiting factor for the cross-coupling reaction of 1,4-NPQ in the initial reaction stages, whereas the birnessite-catalyzed surface reaction acts as the major limiting factor in the later reaction stages. This was explained by the operation of two different reaction mechanisms and reaction products identified by LC-MS/MS.

In this study, a combined media filtration process with micro-flocculation (CMF) was developed, to simultaneously treat particulate and dissolved contaminants in urban road runoff. Dual-size foam glass media with stone and sand layers were applied and the efficiency of road runoff treatment was investigated according to filtration and micro-flocculation under various experimental conditions (stone/sand layer ratio, linear velocity, and coagulant types). Moreover, the removal efficiencies of suspended solids (SS), phosphorus, organic carbon, and heavy metals (Zn, Cu, Pb, Cd) by CMF were evaluated. The removal rate of SS was maintained to be above 84.1% for 1 h filtration by the dual-size foam glass, regardless of increasing pressure. The removal of phosphorus by micro-flocculation was more suitable in alum than ferric due to a higher initial floc growth rate and an increased particle size. The performance of the CMF was significantly improved over media filtration only process (MF) in removing both particulate and dissolved contaminants. The removal efficiency of all particulate pollutants by CMF was found to be more than 90%, and notably, the dissolved phosphorus, which was mostly not removed by MF, was also removed by 97.4%. Meanwhile, the backwash efficiency of CMF was half that of MF. Physical removal mechanisms, such as internal diffusion, dominated MF, whereas chemical removal mechanisms, such as adsorption and surface precipitation, dominated CMF. These results show the potential of the CMF process for the treatment of urban road runoff and identify the removal mechanisms of the filtration process that use micro-flocculation with dual-size foam glass.

The effects of chlorination on 16 humic and fulvic acids (HAs and FAs, respectively) extracted from six different soil samples from Korea and two purchased soil samples (Canadian peat moss, Elliott Silt Loam Soil) were investigated to identify the changes in their structural characteristics and their effects on trihalomethane formation potential (THMFP) and haloacetic acid formation potential. The effect of chlorination was also investigated in fractionated samples (Aldrich HA, F1-F5) based on molecular weight (MW). Total organic carbon (TOC), specific UV absorbance (SUVA), fulvic-like fluorescence (%FLF), terrestrial humic-like fluorescence (%THLF), weight-average molecular weight (MWw), and carbon structures (13C NMR) were measured for each sample before and after chlorination, and factors relating to the chlorination mechanism were examined using principal component analysis (PCA). The results showed that the changes in the structural characteristics and the disinfection by-product formation of chlorinated HA and FA differed critically. For chlorinated HA, TOC and %FLF decreased due to oxidation, whereas %THLF was reduced via incorporation; MW also affected the structural changes and THMFP generation. In the PCA results, high SUVA, low MW, low N/C, and low O groups of aromatic C were associated with high THMFP production in HA, whereas low O groups of aliphatic C in FA were associated with both oxidation and incorporation in terms of THMFP. These results elucidate the mechanisms associated with the effects of chlorination in HA and FA and will support the prediction of THMFP generation in HA and FA based on their specific structural characteristics.

The importance of tributary management is increasing owing to the increase and diversity of pollutant sources in watersheds. In this study, the characteristics of dissolved and particulate contaminants in tributaries of the Anyang watershed, South Korea, were investigated using various analytical indicators (e.g., organic matter, nutrients, and spectroscopic analysis), heat maps, and multivariate analysis techniques, including cluster analysis and principal component analysis (PCA), to evaluate water quality characteristics of the tributaries and major pollutant sources. The influence of particulate matter from each tributary was also evaluated by investigating changes in sediment composition and re-suspended sediment oxygen demand (RSOD) at the mainstream of Anyang. The heat map patterns of the evaluated tributaries reflect sufficiently the characteristics of the pollutant sources, which enables the differentiation of the pollutant source according to the tributary locations and seasons. PCA results showed that water quality and spectral indicators reflected the different characteristics of the tributary; thus, it enabled a more diverse evaluation of tributary depending on pollutant source and season. The tributary watershed showed a high suspended solids (SS) discharge load depending on the watershed area (r = 0.94, p 80%). Our results demonstrate that comprehensive and visual evaluation—including spectral indicators and heat maps analysis—of the tributary characteristics of pollutant sources is an efficient approach of broadening the knowledge of the distribution and effects of particulate matter from tributaries.

In this study, we investigated the photochemical release of dissolved organic matter (DOM) from the particulate organic matter (POM) of soil and litter leaves (broad leaves; coniferous leaves) and compared the releasing characteristics of the DOM using UV-visible and fluorescence spectroscopy. The disinfection by-product formation potential (DBPFP) of the released DOM was also examined. Additional dissolved organic carbon (DOC) was released by UV irradiation for all POM sources (10.58 ± 2.7 mg-C L−1 g−1 for BL, 8.32 ± 2.6 mg-C L−1 g−1 for CL, and 0.20 ± 0.1 mg-C L−1 g−1 for soil). The excitation-emission matrix combined with parallel factor analysis results showed that the photo-released DOM from soil was mainly humic-like components (C1, C3) produced by photodesorption, resulting in high trihalomethane formation potential, while protein-like component (C2) was the major component of the photodissolved DOM from litter leaves, resulting in high haloaceticacid formation potential. Further, DBPFP from soil and litter leaves showed high correlation with humic-like components (C1+C3) and SUVA254, respectively. In conclusion, this study demonstrates that significant amounts of DOM could be released from POM under UV irradiation, although the characteristics and DBP formation of the photo-released DOM were highly dependent upon the POM source.

An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

This study was conducted to develop and validate a more reliable total organic carbon (TOC) analytical procedure for water samples containing suspended solids (SS). The effects of the combined ultrasonic and alkaline pretreatment (CULA) on the TOC measurement were studied in water samples containing SS from three origins (algae, sewage particles, and soil) under different analytical conditions (SS concentration, oxidation methods, and sieve size). The applicability of turbidity as a homogeneity index was also evaluated. With CULA, TOC recovery remained high (&gt, 80%) for SS concentration ranges up to four times larger than ultrasonic pretreatment alone (UL) due to enhanced particulate organic carbon (POC) solubilization, and did not significantly differ depending on the oxidation methods, at low SS concentrations, or with varying sieve sizes. In particular, the turbidity change rate (i.e., NTU5/NTU0) of the pretreated water sample showed a high correlation with TOC precision (r2 = 0.73, p &lt, 0.01), which suggests that turbidity can be used as an indicator of sample homogeneity. A novel TOC analytical procedure is expected to be useful for more accurate assessments of the impact of particulate pollutants on water quality than current methods, and for the analysis of the carbon cycle, including POCs, in the environment.

Orostachys japonicus A. Berger (), known as Wa-song in Korea, has been reported to exert various biological effects, such as anti-tumor, anti-oxidant, and anti-febrile effects. However, the anti-angiogenic effects of O. japonicus extracts remain to be investigated. In the present study, we demonstrated the anti-angiogenic effects of bioconverted O. japonicus extract (BOE) in Ms-1 mouse endothelial cells and compared them with the bioactivities of O. japonicus extract (OE). BOE, but not OE, were found to exert anti-angiogenic effects, including inhibition of cell migration, cell adhesion, tube formation of Ms-1 cells, and blood vessel formation of matrigel plug assay in vivo. Furthermore, protein levels of phosphorylated Src kinase were lower in BOE-treated cells than in OE-treated cells. Treatment with OE or BOE did not influence cell viability during the experimental period. Bioconverted extract of O. japonicus have anti-angiogenic effects in vitro and vivo, but non-bioconverted extract do not. We suggest that these observed anti-angiogenic effects are caused by the changes in the composition of bioactive compounds in the extracts as a result of biological conversion.

In May 2015, Middle East respiratory syndrome coronavirus infection was laboratory confirmed in South Korea. Patients were a man who had visited the Middle East, his wife, and a man who shared a hospital room with the index patient. Rapid laboratory confirmation will facilitate subsequent prevention and control for imported cases.

Highlights • We investigated the prevalence of HRSV during 2008–2013. • Novel HRSV-A ON1 genotype was emerged in August 2011. • After 1 year of emergence in 2012–2013, 94.6% was replaced with novel ON1 genotype. • Evolutionary dynamics also drastically increased in 2011. • The result of epitope prediction shows the possibilities of antigenic variation., Human respiratory syncytial virus (HRSV) is the main cause of severe respiratory illness in young children and elderly people. We investigated the genetic characteristics of the circulating HRSV subgroup A (HRSV-A) to determine the distribution of genotype ON1, which has a 72-nucleotide duplication in attachment G gene. We obtained 456 HRSV-A positive samples between October 2008 and February 2013, which were subjected to sequence analysis. The first ON1 genotype was discovered in August 2011 and 273 samples were identified as ON1 up to February 2013. The prevalence of the ON1 genotype increased rapidly from 17.4% in 2011–2012 to 94.6% in 2012–2013. The mean evolutionary rate of G protein was calculated as 3.275 × 10−3 nucleotide substitution/site/year and several positively selected sites for amino acid substitutions were located in the predicted epitope region. This basic and important information may facilitate a better understanding of HRSV epidemiology and evolution.

Recombinant measles virus nucleoproteins (rMeV N) and fusion (F) proteins were characterized as major antigenic proteins expressed in insect cells mediated by recombinant baculoviruses (rBVs). Band intensities were analyzed by Western blotting to recognize IgG and IgM antibodies against the rMeV N and F proteins in human sera and cerebrospinal fluids (CSFs) from patients with measles infections. Positive results from the blots using the rMeV N were consistent with the results of enzyme-linked immunosorbent assays (ELISAs) in which whole viral proteins were used as antigens. Human sera and CSFs reacted more strongly with the rMeV N than with the rMeV F proteins prepared in an identical expression system. For efficient and reliable purification, ion-exchange chromatography using Source Q anion resin was applied, and high-purity rMeV N protein was harvested. To characterize the similarity with the native viral protein to purified N protein, structural mimicry of purified recombinant proteins with intact rMeV N was shown through transmission electron microscopy, and the truncation and the phosphorylation status of the expressed protein were analyzed. These results suggest that the rMeV N purified by ion-exchange chromatography has features similar to those of naïve N including a self-assembled structure, phosphorylation and antigenic function. Thus, these expression and purification methods can be applied to the large-scale production of the rMeV N, which is essential for the development of new diagnostic tools and vaccines for acute and chronic MeV infections.

Polyvinyl amine (PVAm) hydrogel hollow particles treated by layer-by-layer (LbL) assembly methods were fabricated. The structures of the prepared particles were confirmed through zeta-potential measurements and scanning electron microscopy (SEM). In addition, the loading amount and release rate of Chromotrope 2R (CR) from the prepared particles in each deposition step was investigated. The loading amount had an alternating tendency according to an increase in the layer number. When the outermost layer consisted of poly(sodium 4-styrenesulfonate) (PSS), CR permeation was prevented by ionic repulsion between the PSS and CR. On the other hand, CR easily permeated into the particles when the outermost layer was poly(diallyldimethyl ammonium chloride) (PDDA). The release rate was then changed by ionic interaction between the CR and the cationic polyelectrolyte and by the ionic repulsive force between the CR and the anionic polyelectrolyte. These results indicate that PVAm hydrogel hollow particles treated by the LbL method could potentially be used in the controlled release of water-soluble materials.

Recombinant adeno-associated virus serotype 5 (rAAV5) is considered to be a promising gene transfer vehicle. However, preferential gene delivery to the tumor remains a requirement for cancer treatment. We generated rAAV5 mutants bearing tumor marker-binding peptides and analyzed their properties as viral vectors, as well as their transduction efficiencies and preferential antitumoral potencies. All of the mutants were successfully produced. Transduction analyses showed that rAAV5 mutants harboring tumor-homing peptides, including RGD and TnC, transduced human cancer cells expressing corresponding receptors on their surfaces. RGDS peptides and TnC antibodies significantly suppressed transduction by rAAV5-RGD and rAAV5-TnC. Cytotoxicity was evident upon transfer of HSV-TK to cells by re-targeted rAAV5. These results provide evidence that rAAV5 vectors, genetically armed with tumor-targeting ligands, preferentially infect human cancer cells harboring the corresponding receptors, thereby inducing antitumoral effects. Further optimization of rAAV5 mutant viruses should thus facilitate practical exploitation of these vectors for gene-based cancer treatment.

RNA interference (RNAi) is a 'knock-down' reaction to reduce expression of a specific gene through highly regulated, enzyme-mediated processes. Small interfering RNAs (siRNAs) are RNA molecules that play an effector role in RNAi and can bind the PAZ domains present in Dicer and RISC. We investigated the interaction between the PAZ domain and the siRNA-like duplexes through dissociation molecular dynamics (DMD) simulations. Specifically, we focused on the response of the PAZ domain to various 3'-overhang structures of the siRNA-like duplexes. We found that the siRNA-like duplex with the 3' UU-overhang made relatively more stable complex with the PAZ domain compared to those with 3' CC-, AA-, and GG-overhangs. The siRNA-like duplex with UU-overhang was easily dissociated from the PAZ domain once the structural stability of the complex is impaired. Interestingly, the 3' UU-overhang spent the least time at the periphery region of the binding pocket during the dissociation process, which can be mainly attributable to UU-overhang's smallest number of hydrogen bonds.

The full genome sequence of a Middle East respiratory syndrome coronavirus (MERS-CoV) was identified from cultured and isolated in Vero cells. The viral genome sequence has high similarity to 53 human MERS-CoVs, ranging from 99.5% to 99.8% at the nucleotide level.

There is a paucity of complete genome sequence information for human respiratory syncytial virus (HRSV). To this end, we sequenced the complete genome sequences of HRSV genotype A (HRSV-A/IC688/12) and genotype B (HRSV-B/GW0047/14 and HRSV-B/IC0027/14). This information will increase the understanding of HRSV genetic diversity, evolution, pathogenicity, antigenicity, and transmissibility.

DRG2, a member of the DRG subfamily in the GTP-binding protein superfamily, was identified as a repressed gene product in fibroblasts transformed by SV40. The significance of this down-regulation and the cellular role of DRG2 has not been understood in the past. To investigate the function of DRG2 we made a Jurkat cell line, Jurkat-LNCX2-DRG2, stably transfected with pLNCX2-DRG2 to overexpress human DRG2. Cell cycle distribution analysis revealed an increased accumulation of G(2)/M phase cells in Jurkat-LNCX2-DRG2 cells, indicating a retardation of cell-cycle progression. In addition, an overexpression of DRG2 reduced the sensitivity of Jurkat cells to the mitotic poison nocodazole. Our data suggest that overexpression of DRG2 in Jurkat cells affects genes regulating cell-cycle arrest and apoptosis, and that these molecular changes may be important in the growth or differentiation of cells.

PURPOSE This study has been planned to generate a replication-competent adenovirus which replicates in a cancer cell-specific manner, thus minimizing the side effects and toxicity of cancer gene therapy. MATERIALS AND METHODS we have generated an E1B 19 kD attenuated recombinant adenoviruses, Ad-TERT-delta19 and Ad-mTERT-delta19, which encode E1A gene driven by the wild type hTERT and modified m-hTERT promoter containing additional c-myc and Sp1 binding sites in the backbone of Ad-deltaE1B19. The in vitro efficacy and specificity of the hTERT and m-hTERT promoter have been evaluated by the comparison of viral replication and cytopathic effect in cancer cells and normal cell lines. To assess anti-tumor effect and safety of hTERT or m-hTERT promoter driven replication competent adenoviruses, tumor regression after subcutaneous injection in subcutaneous C33A xenografts and lacZ expression after systemic injection in organs were examined. RESULTS The activation of hTERT or m-hTERT promoter was significantly up-regulated only in hTERT-positive cells, but not in hTERT-negative cells. Moreover, the activity of m-hTERT promoter was substantially increased in hTERT-positive cancer cells, but not in hTERT-negative cells. While Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect in cancer cells only. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. CONCLUSION S: The use of m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of m-hTERT promoter may be a new promising tool for the treatment of human malignancies.

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant protein with posttranslational modification. Recombinant baculoviruses (such as Autographa californica multiple nuclear polyhedrosis virus) are especially useful in producing recombinant proteins and virus-like particles (VLPs) as biodrugs or candidate vaccines for the prevention of serious infectious diseases. However, during the bioprocessing of recombinant proteins in insect cells, baculovirus replication and viral budding are coincident. In some cases, residual baculovirus contaminants remain in the recombinant protein products, even though various purification processes are applied such as ion-exchange chromatography, ultracentrifugation, or gel filtration. To reduce unexpected contamination caused by replication and budding-out of the baculovirus, we designed short interfering (si) RNAs targeting glycoprotein 64 (GP64) or single-stranded DNA-binding protein (DBP) to inhibit baculovirus replication during overexpression of recombinant foreign genes. GP64 is known to be critical both for the entry of virions into cells and for the assembly of the budded virion at the cell surface. DBP is also essential for virus assembly by regulation of the capsid protein P39 and the polyhedrin protein. This study showed that GP64 expression was suppressed by GP64 siRNAs in Western blot experiments, while the expression of recombinant proteins was unaffected. In addition, transfection of GP64 siRNAs and DBP siRNAs reduced the level of baculovirus replication, compared with the treatment with scrambled siRNAs. However, DBP siRNA also suppressed the expression of recombinant proteins. In conclusion, our GP64 siRNAs showed that an interfering RNA system, such as siRNAs and short hairpin (sh) RNAs, can be applicable to reduce baculovirus contaminants during the bioprocessing of recombinant proteins in insect cells. Further investigation should be carried out to establish transformed insect cell lines with stable expression of corresponding interfering RNAs.

Despite the well-documented advantages of the recombinant adeno-associated virus (rAAV) as a gene delivery vehicle, including its non-pathogenic and long-term therapeutic gene expression, there have been very limited studies on its potential for producing persistent anti-tumor effects, particularly in vivo. To address this issue, we constructed rAAV vectors encoding herpes simplex virus 1-thymidine kinase (HSV-TK) or its mutant form (sc39TK) as therapeutic genes, and GFP as a control gene. Effective rAAV-mediated gene delivery was readily observed in human cancer cells using immunocytochemistry and Western blotting. Cell survival analysis following prodrug ganciclovir treatment implied that both preferential and superior cytotoxicity was achieved by rAAV-sc39TK introduction. Persistent anti-tumor effects in vivo were investigated in Balb/c nude mice bearing human cancer cells treated with either rAAV-sc39TK or -GFP. Severe tumor growth inhibition was clearly observed only in the case of sc39TK with ganciclovir treatment. Non-invasive micro-PET imaging using 18F-FHBG directly correlated with persistent anti-tumor effects by sc39TK. Therefore, the present study provides evidence that rAAV-mediated persistent therapeutic gene expression can occur, resulting in long-term anti-tumor activities and that these events can be readily monitored using micro-PET imaging.

The feasibility of various self-complementary AAV (scAAV) serotypes as efficient gene delivery vehicles in human cancer cells was evaluated. To dissect the transduction charac- teristics, we infected a variety of human cancer cells with scAAV1-6 or scAAV8 expressing GFP. scAAV2 led to the best transduction efficiency with nearly complete transgene expression at 1000 MOI in most cancer cells, regardless of cell/tissue origins. scAAV5 could also induce effective gene expression, even though gene transfer potency by scAAV5 was poorer than that by scAAV2. Substantial portion of trans- gene expression lasted over a month following gene delivery by both scAAV 2 and scAAV5, indicating that long-term gene expression can occur. Moreover, co-infection of scAAV2 and scAAV5 can induce simultaneous transgene expressions introduced via each vector. Thus, the current study provide evidence that scAAV2 and scAAV5 vectors are excellent gene transfer tools in a wide variety of human cancer cells, independently driving persistent transgene expression.

Although the human telomerase reverse transcriptase (hTERT) promoter can regulate cancer-specific genes, it is generally too weak to be effective. We therefore attempted to improve the potency of synthetic hTERT promoters by fusing the core element (E) of the hTERT promoter (H) and the tripartite leader sequence (T) from human adenovirus 5 in a combinatorial manner. To determine the potential as cancer-specific promoters, we measured luciferase activity driven by the chimeric hTERT promoters in human cancer cells. Among various constructs, the E3-H-T promoter induced the strongest luciferase activity in all the tested cancer cells. SK-Hep1 and Hela cells experienced 1000- and 11-fold higher expression than the basic hTERT promoter, respectively. Relative to the SV40 universal promoter, the E3-H-T promoter led to higher levels of gene expression. Using EMSA, we found that the hTERT enhancer region was specifically bound to c-Myc and Sp1. Thus, the data suggest that the E3-H-T promoter with up-regulated cancer-specific gene expression could be useful in cancer gene therapy.

The complete genome sequence of human respiratory syncytial virus genotype A (HRSV-A) with a 72-nucleotide duplication in the C-terminal part of the attachment protein G gene was determined and analyzed. The genome was 15,277 bp in length, and 0.46 to 6.03% variations were identified at the nucleotide level compared with the previously reported complete genome of HRSV-A. Characterization of the genome will improve understanding of the diversity of the HRSV-A major antigens and enable an in-depth analysis of its genetics.