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Black tiger shrimp ( Penaeus monodon ) is an economically important species for aquaculture in Vietnam; however, very little information is available regarding its transcriptome. In this study, we sequenced four transcriptome libraries from mRNA of heart, muscle, hepatopancreas, and eyestalk tissues of P. monodon individuals using the Illumina MiSeq® platform, yielding 78,575,346 raw paired-end reads. After quality control, a total of 62,327,726 paired-end reads were de novo assembled into 69,089 unigenes with an average size of 447.89 bp, N50 of 481 bp, and transcriptome size of 30.94 Mb. Comparisons of the assembled unigenes against five public protein databases identified 165 unigenes related to growth and development, and expression analysis revealed that 14,229 unigenes were differentially expressed among tissues. In addition, we detected 12,768 simple sequence repeats (SSRs) in 11,173 unigenes. This work provides candidate genes for trait mapping, marker-assisted breeding, genetic studies and functional genome annotation of P. monodon . Statement of relevance This paper is relevant for the Shrimp Industry.

This article reports on the ability of yeast Trichosporon asahii B1 biofilm-associated cells, compared with that of planktonic cells, to transform sec-hexylbenzene and its metabolites. This B1 strain was isolated from a petroleum-polluted sediment collected in the QuangNinh coastal zones in Vietnam, and it can transform the branched aromatic hydrocarbons into a type of forming biofilm (pellicle) more efficiency than that the planktonic forms can. In the biofilm cultivation, seven metabolites, including acetophenone, benzoic acid, 2,3-dihydroxybenzoic acid, β-methylcinnamic acid, 2-phenylpropionic acid, 3-phenylbutyric acid, and 5-phenylhexanoic acid were extracted by ethyl acetate and analyzed by HPLC and GC-MS. In contrast, in the planktonic cultivation, only three of these intermediates were found. An individual metabolite was independently used as an initial substrate to prove its degradation by biofilm and planktonic types. The degradation of these products indicated that their inoculation with B1 biofilms was indeed higher than that observed in their inoculation with B1 planktonic cells. This is the first report on the degradation of sec-hexylbenzene and its metabolites by a biofilm-forming Trichosporon asahii strain. These results enhance our understanding of the degradation of branched-side-chain alkylbenzenes by T. asahii B1 biofilms and give a new insight into the potential role of biofilms formed by such species in the bioremediation of other recalcitrant aromatic compounds.

World production of farmed crustaceans was 7.8 million tons in 2016. While only making up approximately 10% of world aquaculture production, crustaceans are generally high-value species and can earn significant export income for producing countries. Viet Nam is a major seafood producing country earning USD 7.3 billion in 2016 in export income with shrimp as a major commodity. However, there is a general lack of genomic resources available for shrimp species, which is challenging to obtain due to the need to deal with large repetitive genomes, which characterize many decapod crustaceans. The first tiger prawn (P. monodon) genome assembly was assembled in 2016 using the standard Illumina PCR-based pair-end reads and a computationally-efficient but relatively suboptimal assembler, SOAPdenovo v2. As a result, the current P. monodon draft genome is highly fragmented (> 2 million scaffolds with N50 length of

Microalgae are the most promising sources of protein, which have high potential due to their high-value protein content. Conventional methods of protein harnessing required multiple steps, and they are generally complex, time consuming, and expensive. Currently, the study of integration methods for microalgae cell disruption and protein recovery process as a single-step process is attracting considerable interest. This study aims to investigate the novel approach of integration method of electrolysis and liquid biphasic flotation for protein extraction from wet biomass of Chlorella sorokiniana CY-1 and obtaining the optimal operating conditions for the protein extraction. The optimized conditions were found at 60% (v/v) of 1-propanol as top phase, 250 g/L of dipotassium hydrogen phosphate as bottom phase, crude microalgae loading of 0.1 g, air flowrate of 150 cc/min, flotation time of 10 min, voltage of 20 V and electrode’s tip touching the top phase of LBEF. The protein recovery and separation efficiency after optimization were 23.4106 ± 1.2514% and 173.0870 ± 4.4752%, respectively. Comparison for LBEF with and without the aid of electric supply was also conducted, and it was found that with the aid of electrolysis, the protein recovery and separation efficiency increased compared to the LBEF without electrolysis. This novel approach minimizes the steps for overall protein recovery from microalgae, time consumption, and cost of operation, which is beneficial in bioprocessing industry.

In bio-based fermentation, the overall bioprocess efficiency is significantly affected by the metabolic burden associated with the expression of complete biosynthetic pathway as well as precursor and cofactor generating enzymes into a single microbial cell. To attenuate such burden by compartmentalizing the enzyme expression, recently synthetic biologists have used coculture or poly-culture techniques for biomolecules synthesis. In this paper, coculture system of two metabolically engineered Escherichia coli populations were employed which comprises upstream module expressing two enzymes converting para-coumaric acid into resveratrol and the downstream module expressing glucosyltransferase to convert the resveratrol into its glucosidated forms; polydatin and resveratroloside. Upon optimization of the initial inoculum ratio of two E. coli populations, 92 mg resveratrol glucosides/L (236 µM) was produced i.e. achieving 84% bioconversion from 280 µM of p-coumaric acid in 60 h by 3 L fed batch fermentor. This is the report of applying coculture system to produce resveratrol glucosides by expressing the aglycone formation pathway and sugar dependent pathway into two different cells.

The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig-fertilization medium was superior medium to Tyrode's albumin lactate pyruvate-polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 106 sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus-oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig-fertilization medium from 2 to 5 mmol/L and the prolongation of the co-culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig-fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility.

Sacbrood virus (SBV) is one of the most common viral infections of honeybees. The entire genome sequence for nine SBV infecting honeybees, Apis cerana and Apis mellifera, in Vietnam, namely AcSBV-Viet1, AcSBV-Viet2, AcSBV-Viet3, AmSBV-Viet4, AcSBV-Viet5, AmSBV-Viet6, AcSBV-Viet7, AcSBV-Viet8, and AcSBV-Viet9, was determined. These sequences were aligned with seven previously reported complete genome sequences of SBV from other countries, and various genomic regions were compared. The Vietnamese SBVs (VN-SBVs) shared 91–99% identity with each other, and shared 89–94% identity with strains from other countries. The open reading frames (ORFs) of the VN-SBV genomes differed greatly from those of SBVs from other countries, especially in their VP1 sequences. The AmSBV-Viet6 and AcSBV-Viet9 genome encodes 17 more amino acids within this region than the other VN-SBVs. In a phylogenetic analysis, the strains AmSBV-Viet4, AcSBV-Viet2, and AcSBV-Viet3 were clustered in group with AmSBV-UK, AmSBV-Kor21, and AmSBV-Kor19 strains. Whereas, the strains AmSBV-Viet6 and AcSBV-Viet7 clustered separately with the AcSBV strains from Korea and AcSBV-VietSBM2. And the strains AcSBV-Viet8, AcSBV-Viet1, AcSBV-Viet5, and AcSBV-Viet9 clustered with the AcSBV-India, AcSBV-Kor and AcSBV-VietSBM2. In a Simplot graph, the VN-SBVs diverged stronger in their ORF regions than in their 5′ or 3′ untranslated regions. The VN-SBVs possess genetic characteristics which are more similar to the Asian AcSBV strains than to AmSBV-UK strain. Taken together, our data indicate that host specificity, geographic distance, and viral cross-infections between different bee species may explain the genetic diversity among the VN-SBVs in A. cerana and A. mellifera and other SBV strains.

Many studies have comprehensively examined the venom ofOphiophagus hannahsnake. Its venom comprises different compounds exhibiting a wide range of pharmacological activities. In this investigation, four peptide fractions (PFs), ranging from 3 kDa to 10 kDa, isolated from the Vietnamese snake venom ofO. hannahwere separated by HPLC and investigated for their inhibitory activity on adipogenesis in 3T3-L1 adipocytes. The most effective PF was then further purified, generating two peptides, pOh1 and pOh2. Upon investigation of these two peptides on 3T3-L1 adipocytes, it was revealed that, at 10 μg/mL, pOh2 was able to inhibit the lipid accumulation in 3T3-L1 adipocytes by up to 56%, without affecting cell viability. Furthermore, the pOh2 downregulated the gene expression of important transcription factors C/EBP-αand PPAR-γ. In addition, aP2 and GPDH adipocyte-specific markers were also significantly reduced compared to untreated differentiated cells. Taken together, pOh2 inhibited the expression of key transcription factors C/EBP-αand PPAR-γand their target genes, aP2 and GPDH, thereby blocking the adipocyte differentiation. In conclusion, this novel class of peptide might have potential for in vivo antiobesity effects.

Deformed wing virus (DWV) is one of the most common viral infection in honeybees. Phylogenetic trees were constructed for 16 partial nucleotide sequences of the structural polyprotein region and the RNA helicase region of South Korean DWVs. The sequences were compared with 10 previously reported DWV sequences from different countries and the sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus-1 (VDV-1). The phylogeny based on these two regions, the Korean DWV genomes were highly conserved with 95-100% identity, while they also shared 93-97% similarity with genotypes from other countries, although they formed a separate cluster. To investigate this phenomenon in more detail, the complete DWV genome sequences of Korea-1 and Korea-2 were determined and aligned with six previously reported complete DWV genome sequences from different countries, as well as KGV and VDV-1, and a phylogenetic tree was constructed. The two Korean DWVs shared 96.4% similarity. Interestingly, the Korea-2 genome was more similar to the USA (96.5%) genome than the Korea-1. The Korean genotypes highly conserved with USA (96%) but low similarity with the United Kingdom3 (UK3) genome (89%). The end of the 5' untranslated region (UTR), the start of the open reading frame (ORF) region, and the 3' UTR were variable and contained several substitutions/transitions. This phenomenon may be explained by intramolecular recombination between the Korean and other DWV genotypes.

Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries.

The emergence of highly pathogenic avian influenza (HPAI) H5N1 viruses and the risk of a human pandemic have highlighted the need for advance stockpiling of vaccine. Current vaccines may be sub-optimally matched to the actual pandemic virus due to the rapid dissemination and ongoing evolution of avian H5N1 viruses. We report here the evaluation of efficacy of NIBRG-14 vaccine (clade 1 A/Vietnam/1194/2004) against the H5N1 HPAI virus strains circulating in Vietnam. The birds were either vaccinated with a single or booster dose of vaccine by subcutaneous injection; then challenged with three H5N1 HPAI viruses (clade 1, clade 2.3.2.1a and clade 2.3.2.1b) at day 21 post-vaccination (p. v.). The results showed that NIBRG-14 vaccine protected birds from clade 1 and clade 2.3.2.1a infections. Notably, we observed that NIGRG-14 vaccine did not confer protection against clade 2.3.2.1b challenge virus. To get new insights of how H5N1 clade 2.3.2.1b (A/Duck/QuangNgai/1037/11) virus can escape from the host immune response induced by the vaccine, we further analyzed the HA gene - a key virulence determinant of the virus. Amino acid sequence analysis indicated that this virus contained the sequence SPQRERRRK-R/G at the cleavage site in the HA molecule, indicating its high virulence. Additionally, we identified numerous mutations with amino acid substitutions in the hemagglutinin: M226I, I239S located at N-link glycosylation site and 2H, 45N, 53K 120D, 133A and 14N mutations at antigenic site, which can affect receptor specificity as well as viral pathogenicity. Notably, I239S and S133A mutations are unique to A/Duck/QuangNgai/1037/2011, suggesting that it may involve in the virus' ability to evade the host immune system. Taken together, phylogenetic analysis showed that continual mutations in the HA gene may have generated a novel antigenic strain and that probably changed the virulence of the virus and made the H5N1 clade 2.3.2.1b resistant to NIBRG-14 vaccine.

Avian influenza virus vaccines produced in oil-emulsified inactivated form with antigen content of at least 160 hemagglutinin units (HAU) induced immunity in birds. However, in addition to enhancing the effect of the adjuvant(s), other additional supplemented biological compounds included in inactivated vaccines could produce higher levels of antibody. We examined in chickens, Vietnamese ducks, and muscovy ducks the adjuvant effect of Sophy β-glucan (SBG), a β-1,3-1,6 glucan produced by the black yeast Aureobasidium pollulans strain AF0-202, when administered with an avian influenza H5 subtype vaccine. In Experiment 1, 40 chickens (ISA Brown hybrid), allocated to four groups of ten each, were immunized with Oil-H5N1(VN), Oil-H5N1(CN), Oil-H5N2(CN), and saline (control group), respectively. In Experiment 2, chickens (ISA Brown hybrid), muscovy ducks (French hybrid), and Vietnamese ducks (indigenous Vietnamese) were used to further assess the effect of SBG on immunogenicity of the Oil-H5N1(VN) Vietnamese vaccine. ELISA and hemagglutination inhibition (HI) assays were used to assess the antibody response. The H5 subtype vaccines initiated significantly higher immune responses in the animals dosed with SBG, with 1.0-1.5 log2 higher HI titers and 10-20% ELISA seroconversion, compared with those not dosed with β-glucan. Notably, some of the animals dosed with SBG induced HI titers higher than 9.0 log2 following boosting immunization. Taken together, our serial studies indicated that SBG is a potential effector, such as enhancing the immune response to the H5 vaccines tested.

To understand the mechanisms underlying 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE]–induced angiogenesis, we studied the role of Egr-1. 15(S)-HETE induced Egr-1 expression in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Blockade of Egr-1 via forced expression of its dominant-negative mutant attenuated 15(S)-HETE–induced HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. 15(S)-HETE–induced Egr-1 expression requires Src activation. In addition, adenovirus-mediated expression of dominant-negative mutant of Src blocked 15(S)-HETE's effects on migration and tube formation of HDMVECs and Matrigel plug angiogenesis. 15(S)-HETE induced fibroblast growth factor-2 (FGF-2) expression rapidly via Src-mediated production of Egr-1. Cloning and mutational analysis of FGF-2 promoter revealed that Egr-1 binding site proximal to transcription start site is required for 15(S)-HETE–induced FGF-2 expression. Neutralizing antibody-mediated suppression of FGF-2 function also attenuated the effects of 15(S)-HETE on HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. Furthermore, in contrast to wild-type mice, 12/15-LOX−/− mice exhibited decreased Matrigel plug angiogenesis in response to AA, which was rescued by 15(S)-HETE. On the basis of these observations, we conclude that 15(S)-HETE–induced angiogenesis requires Src-mediated Egr-1–dependent rapid induction of FGF-2. These findings may suggest that 15(S)-HETE could be a potential endogenous regulator of pathologic angiogenesis associated with atherosclerosis and restenosis.

To understand the role of human 15-lipoxygenase 1 (15-LOX1) in vascular wall remodeling, we have studied the effect of the major 15-LOX1 metabolite of arachidonic acid, 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), on vascular smooth muscle cell (VSMC) migration both in vitro and in vivo. Among 5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, 15(S)-HETE potentially stimulated more vascular smooth muscle cell (VSMC) migration. In addition, 15(S)-HETE-induced VSMC migration was dependent on Src-mediated activation of signal transducer and activator of transcription-3 (STAT-3). 15(S)-HETE also induced monocyte chemoattractant protein-1 (MCP-1) expression via Src-STAT-3 signaling, and neutralizing anti-MCP-1 antibodies completely negated 15(S)-HETE-induced VSMC migration. Cloning and characterization of a 2.6-kb MCP-1 promoter revealed the presence of four putative STAT-binding sites, and the site that is proximal to the transcription start site was found to be essential for 15(S)-HETE-induced Src-STAT-3-mediated MCP-1 expression. Rat carotid arteries that were subjected to balloon injury and transduced with Ad-15-LOX1 upon exposure to [(3)H]arachidonic acid ex vivo produced 15-HETE as a major eicosanoid and enhanced balloon injury-induced expression of MCP-1 in smooth muscle cells in Src and STAT-3-dependent manner in vivo. Adenovirus-mediated delivery of 15-LOX1 into rat carotid artery also led to recruitment and homing of macrophages to medial region in response to injury. In addition, transduction of Ad-15-LOX1 into arteries enhanced balloon injury-induced smooth muscle cell migration from media to intima and neointima formation. These results show for the first time that 15-LOX1-15(S)-HETE axis plays a major role in vascular wall remodeling after balloon angioplasty.

Gerbils (Meriones unguiculatus) were inoculated intraperitoneally (i.p.) with Neospora caninum tachyzoites to examine parasite distribution and histological lesions at different time points over a 9-day period of infection. Gerbils were sacrificed 12 h post-infection (PI), then daily intervals up to day 9 PI. The parasite was detected by PCR assay targeting the Nc5 sequence of N. caninum. The parasite was not found in any organs until day 5 PI, however, from day 8 PI onwards, they were detected in all the organs examined as demonstrated by PCR. The first target organs in acute N. caninum infection were liver, spleen, and kidney, but not the blood as was expected. Histologic lesions were detected in the liver and spleen only, no lesions were found in other organs examined until the end of the experiment. Notably, the focal miliary hepatitis was observed in the liver of infected gerbils just after 1 day post-inoculation, whereas splenic lesions were not found until day 5 PI. These results reinforce the applicability of gerbils as a suitable model of acute neosporosis and provide new insights into the response of gerbils to N. caninum intraperitoneal infection.

Interconversion between tachyzoites and bradyzoites of Neospora caninum plays a pivotal role in transmission of the parasite. Although significant efforts have been made toward understanding the mechanisms that trigger and control stage conversion of the parasite, little is known about this process. We used annealing control primer (ACP)-based polymerase chain reaction (PCR) technique to characterize the differences in transcription between tachyzoites and bradyzoites of N. caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by treatment of infected cultures with 70 microM sodium nitroprusside. Subsequently, the gene expression profiles of the tachyzoites and bradyzoites were analyzed through comparison of the level of messenger RNA expression. ACP-based PCR revealed 85 amplicons that were consistently differentially expressed between tachyzoite and bradyzoite stages. Of the 85 differentially expressed transcripts identified, ten were cloned into Topo TA cloning vector, sequenced, and further analyzed by the Basic Local Alignment Search Tool. These differentially expressed transcripts include a combination of known genes and as yet unidentified genes. The present work provides candidate genes for further investigation on molecular basis of stage conversion from tachyzoites to bradyzoites of N. caninum.

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.

The E3L gene is essential for pathogenesis in vaccinia virus. The E3L gene product consists of an N-terminal Zα domain and a C-terminal double-stranded RNA (dsRNA) binding domain; the left-handed Z-DNA-binding activity of the Zα domain of E3L is required for viral pathogenicity in mice. E3L is highly conserved among poxviruses, including the smallpox virus, and it is likely that the orthologous Zα domains play similar roles. To better understand the biological function of E3L proteins, we have investigated the Z-DNA-binding behavior of five representative Zα domains from poxviruses. Using surface plasmon resonance (SPR), we have demonstrated that these viral Zα domains bind Z-DNA tightly. Ability of Zα[subscript E3L] converting B-DNA to Z-DNA was measured by circular dichroism (CD). The extents to which these Zαs can stabilize Z-DNA vary considerably. Mutational studies demonstrate that residues in the loop of the β-wing play an important role in this stabilization. Notably the Zα domain of vaccinia E3L acquires ability to convert B-DNA to Z-DNA by mutating amino acid residues in this region. Differences in the host cells of the various poxviruses may require different abilities to stabilize Z-DNA; this may be reflected in the observed differences in behavior in these Zα proteins., Korean Science and Engineering Foundation (National Research Laboratory Program (NRL-2006-02287)), Korean Science and Engineering Foundation (Ubiquitome Research Program (M10533010002-06N3301-00210)), Korean Science and Engineering Foundation (21C Frontier Functional Proteomics Program (FPR06B2-120)), National Institutes of Health (U.S.), Ellison Medical Foundation, Korea (South). Ministry of Science and Technology (National Laboratory program (NRL-2006-02287))

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea. *E-mail: kkim@med.skku.ac.kr Sungkyunkwan Advanced Institute of Nanotechnology, Sungkyunkwan University, Suwon 440-746, Korea Department of Chemistry, School of Natural Sciences, Sungkyunkwan University, Suwon 440-746, Korea *E-mail: ygkimmit@skku.edu Received May 8, 2006

Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97–100 % identity. They also showed 92–99 % similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4–6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92 %) with Hungary10, but low similarity (86 %) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5′-UTR, the intergenic region, and the capsid regions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5′-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.

Ticks are vectors of various pathogens that affect humans and animals throughout the world. Anaplasma bovis is one of the most important tick-borne pathogens that cause cattle diseases but there is still very little information available about this agent in Korea. In the present study, 535 Haemaphysalis longicornis tick pools were analyzed from grazing cattle in five Korean provinces. A. bovis was detected in 50 (9.3%) of 535 tick pools using 16S rRNA-based PCR. A. bovis infections were detected for the first time in ticks feeding on cattle in Chungbuk, Geongbuk, and Jeonbuk provinces in Korea. The 50 positive PCR products were sequenced successfully and compared with sequences in GenBank. Phylogenetic analysis of the Korean isolates classified them into four genotypes with nucleotide sequence identities of 99.4–100%. Two of the four genotypes had high similarity (99.8–100%) with known sequences. The other two genotypes have never been identified.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Thrombin, a G protein-coupled receptor agonist, induced a biphasic expression of cyclin D1 in primary vascular smooth muscle cells. Although both phases of cyclin D1 expression require binding of the newly identified cooperative complex, NFATc1·STAT-3, to its promoter, the second phase, which is more robust, depends on NFATc1-mediated recruitment of p300 onto the complex and the subsequent acetylation of STAT-3. In addition, STAT-3 is tyrosine-phosphorylated in a biphasic manner, and the late phase requires NFATc1-mediated p300-dependent acetylation. Furthermore, interference with acetylation of STAT-3 by overexpression of acetylation null STAT-3 mutant led to the loss of the late phase of cyclin D1 expression. EMSA analysis and reporter gene assays revealed that NFATc1·STAT-3 complex binding to the cyclin D1 promoter led to an enhanceosome formation and facilitated cyclin D1 expression. In the early phase of its expression, cyclin D1 is localized mostly in the cytoplasm and influenced cell migration. However, during the late and robust phase of its expression, cyclin D1 is translocated to the nucleus and directed cell proliferation. Together, these results demonstrate for the first time that the dual function of cyclin D1 in cell migration and proliferation is temperospatially separated by its biphasic expression, which is mediated by cooperative interactions between NFATc1 and STAT-3.

To understand the mechanisms by which 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) activates signal transducer and activator of transcription 3 (STAT3), we studied the role of epidermal growth factor receptor (EGFR). 15(S)-HETE stimulated tyrosine phosphorylation of EGFR in a time-dependent manner in vascular smooth muscle cells (VSMCs). Interference with EGFR activation blocked 15(S)-HETE-induced Src and STAT3 tyrosine phosphorylation, monocyte chemoattractant protein-1 (MCP-1) expression and VSMC migration. 15(S)-HETE also induced tyrosine phosphorylation of Janus kinase 2 (Jak2) in VSMCs, and its inhibition substantially reduced STAT3 phosphorylation, MCP-1 expression, and VSMC migration. In addition, Src formed a complex with EGFR and Jak2, and its inhibition completely blocked Jak2 and STAT3 phosphorylation, MCP-1 expression, and VSMC migration. 15(S)-HETE induced the production of H(2)O(2) via an NADPH oxidase-dependent manner and its scavengers, N-acetyl cysteine (NAC) and catalase suppressed 15(S)-HETE-stimulated EGFR, Src, Jak2, and STAT3 phosphorylation and MCP-1 expression. Balloon injury (BI) induced EGFR, Src, Jak2, and STAT3 phosphorylation, and inhibition of these signaling molecules attenuated BI-induced MCP-1 expression and smooth muscle cell migration from the medial to the luminal surface resulting in reduced neointima formation. In addition, inhibition of EGFR blocked BI-induced Src, Jak2, and STAT3 phosphorylation. Similarly, interference with Src activation suppressed BI-induced Jak2 and STAT3 phosphorylation. Furthermore, adenovirus-mediated expression of dnJak2 also blocked BI-induced STAT3 phosphorylation. Consistent with the effects of 15(S)-HETE on the activation of EGFR-Src-Jak2-STAT3 signaling in VSMCs in vitro, adenovirus-mediated expression of 15-lipoxygenase 1 (15-Lox1) enhanced BI-induced EGFR, Src, Jak2, and STAT3 phosphorylation leading to enhanced MCP-1 expression in vivo. Blockade of Src or Jak2 suppressed BI-induced 15-Lox1-enhanced STAT3 phosphorylation, MCP-1 expression, and neointima formation. In addition, whereas dominant negative Src blocked BI-induced 15-Lox1-enhanced Jak2 phosphorylation, dnJak2 had no effect on Src phosphorylation. Together, these observations demonstrate for the first time that the 15-Lox1-15(S)-HETE axis activates EGFR via redox-sensitive manner, which in turn mediates Src-Jak2-STAT3-dependent MCP-1 expression leading to vascular wall remodeling.

A one-step RT-PCR method using newly designed primers VN-VP1F/VN-VP1R targeting the full VP1 capsid protein-coding gene, combined with direct sequencing of its PCR product, has been developed successfully for universal detection and characterization of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The one-step RT-PCR amplified 821-bp dsDNA products covering the entire VP1 gene of FMDV serotypes O, A, and Asia 1. The obtained dsDNA products were suitable for direct sequencing, cloning, and other molecular epidemiology studies of Vietnamese FMDV strains, which eliminated the need for cell culture and virus purification. This one-step RT-PCR system was applied to detect and characterize 55 field FMDV strains, including 34 serotype O, 17 serotype A, and 4 serotype Asia 1 isolates collected from endemic outbreaks in Vietnam from 2005 to 2010. Interestingly, the PCR products obtained from the present PCR method could be used as DNA templates for the second PCR typing method using serotypes O, A, and Asia 1-specific primers (Le et al., 2011). The use of the second PCR amplification increased markedly the sensitivity of the test for FMDV detection. The present RT-PCR method promises to be an effective tool for molecular epidemiological studies of FMD in Vietnam.

To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)-induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Inhibition of MMP-2 activity or depletion of its levels attenuated 15(S)-HETE-induced HDMVEC migration, tube formation, and Matrigel plug angiogenesis. 15(S)-HETE also induced Fra-1 and c-Jun expression in a Rac1-MEK1-JNK1-dependent manner. In addition, 15(S)-HETE-induced MMP-2 expression and activity were mediated by Rac1-MEK1-JNK1-dependent activation of AP-1 (Fra-1/c-Jun). Cloning and site-directed mutagenesis of MMP-2 promoter revealed that AP-1 site proximal to the transcriptional start site is required for 15(S)-HETE-induced MMP-2 expression, and Fra-1 and c-Jun are the essential components of AP-1 that bind to MMP-2 promoter in response to 15(S)-HETE. Hind limb ischemia led to an increase in MEK1 and JNK1 activation and Fra-1, c-Jun, and MMP-2 expression resulting in enhanced neovascularization and recovery of blood perfusion in wild-type mice as compared with 12/15-Lox(-/-) mice. Together, these results provide the first direct evidence for a role of 12/15-Lox-12/15(S)-HETE axis in the regulation of ischemia-induced angiogenesis.

Platelet-derived growth factor BB induced cyclin D1 expression in a time- and nuclear factor of activated T cells (NFAT)-dependent manner in human aortic smooth muscle cells (HASMCs), and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G(1) to S phase. Selective inhibition of NFATc1 by its small interfering RNA also blocked HASMC proliferation and migration. Characterization of the cyclin D1 promoter revealed the presence of several NFAT binding sites, and the site at nucleotide -1333 was found to be sufficient in mediating platelet-derived growth factor BB-induced cyclin D1 promoter-luciferase reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in an NFATc1-dependent manner. Balloon injury-induced cyclin D1-CDK4 activity requires NFAT activation, and adenovirus-mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury-induced vascular wall remodeling events, including smooth muscle cell migration from the medial to luminal region, their proliferation in the intimal region, and neointima formation. Together, these results provide more mechanistic evidence for the role of NFATs, particularly NFATc1, in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the renarrowing of artery after angioplasty.

A conserved feature of poxviruses is a protein, well characterized as E3L in vaccinia virus, that confers IFN resistance on the virus. This protein comprises two domains, an N-terminal Z-DNA-binding protein domain (Zα) and a C-terminal double-stranded RNA-binding domain. Both are required for pathogenicity of vaccinia virus in mice infected by intracranial injection. Here, we describe the crystal structure of the Zα domain from the E3L-like protein of Yaba-like disease virus, a Yatapoxvirus, in a complex with Z-DNA, solved at a 2.0-Å resolution. The DNA contacting surface of Yaba-like disease virus Zα E3L closely resembles that of other structurally defined members of the Zα family, although some variability exists in the β-hairpin region. In contrast to the Z-DNA-contacting surface, the nonbinding surface of members of the Zα family are unrelated; this surface may effect protein-specific interactions. The presence of the conserved and tailored Z-DNA-binding surface, which interacts specifically with the zigzag backbone and syn base diagnostic of the Z-form, reinforces the importance to poxvirus infection of the ability of this protein to recognize the Z-conformation.