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Background Ginsenoside Rh2, a major saponin derivative in ginseng extract, is recognized for its anticancer activities. Compared to coding genes, studies on long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) that are regulated by Rh2 in cancer cells, especially on competitive endogenous RNA (ceRNA) are sparse. Methods LncRNAs whose promoter DNA methylation level was significantly altered by Rh2 were screened from methylation array data. The effect of STXBP5-AS1, miR-4425, and RNF217 on the proliferation and apoptosis of MCF-7 breast cancer cells was monitored in the presence of Rh2 after deregulating the corresponding gene. The ceRNA relationship between STXBP5-AS1 and miR-4425 was examined by measuring the luciferase activity of a recombinant luciferase/STXBP5-AS1 plasmid construct in the presence of mimic miR-4425. Results Inhibition of STXBP5-AS1 decreased apoptosis but stimulated growth of the MCF-7 cells, suggesting tumor-suppressive activity of the lncRNA. MiR-4425 was identified to have a binding site on STXBP5-AS1 and proven to be downregulated by STXBP5-AS1 as well as by Rh2. In contrast to STXBP5-AS1, miR-4425 showed pro-proliferation activity by inducing a decrease in apoptosis but increased growth of the MCF-7 cells. MiR-4425 decreased luciferase activity from the luciferase/STXBP5-AS1 construct by 26%. Screening the target genes of miR-4425 and Rh2 revealed that Rh2, STXBP5-AS1, and miR-4425 consistently regulated tumor suppressor RNF217 at both the RNA and protein level. Conclusion LncRNA STXBP5-AS1 is upregulated by Rh2 via promoter hypomethylation and acts as a ceRNA, sponging the oncogenic miR-4425. Therefore, Rh2 controls the STXBP5-AS1/miR-4425/RNF217 axis to suppress breast cancer cell growth., Graphical abstract Image 1

International audience; Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available.

Background: Tamoxifen (tam) is widely used to treat estrogen-positive breast cancer. However, cancer recurrence after chemotherapy remains a major obstacle to achieve good patient prognoses. In this study, we aimed to identify genes responsible for epigenetic regulation of tam resistance in breast cancer. Methods: Methylation microarray data were analyzed to screen highly hypomethylated genes in tam resistant (tamR) breast cancer cells. Quantitative RT-PCR, Western blot analysis, and immunohistochemical staining were used to quantify expression levels of genes in cultured cells and cancer tissues. Effects of matrix metalloproteinase-1 (MMP1) expression on cancer cell growth and drug resistance were examined through colony formation assays and flow cytometry. Xenografted mice were generated to investigate the effects of MMP1 on drug resistance in vivo. Results: MMP1 was found to be hypomethylated and overexpressed in tamR MCF-7 (MCF-7/tamR) cells and in tamR breast cancer tissues. Methylation was found to be inversely associated with MMP1 expression level in breast cancer tissues, and patients with lower MMP1 expression exhibited a better prognosis for survival. Downregulating MMP1 using shRNA induced tam sensitivity in MCF-7/tamR cells along with increased apoptosis. The xenografted MCF-7/tamR cells that stably expressed short hairpin RNA (shRNA) against MMP1 exhibited retarded tumor growth compared to that in cells expressing the control shRNA, which was further suppressed by tam. Conclusions: MMP1 can be upregulated through promoter hypomethylation in tamR breast cancer, functioning as a resistance driver gene. MMP1 can be a potential target to suppress tamR to achieve better prognoses of breast cancer patients.

Fusarium graminearum is the causal agent of Fusarium head blight in cereal crops. As in other filamentous ascomycetes, F. graminearum contains genes encoding putative hydrophobins, which are small secreted amphiphilic proteins with eight conserved cysteine residues. Here, we investigated the roles of all five hydrophobin genes (designated FgHyd1, FgHyd2, FgHyd3, FgHyd4, and FgHyd5) in various mycological traits of F. graminearum. Gene expression analyses revealed that the five FgHyd genes, all of which were under the control of G protein signaling or velvet complex proteins, were differentially expressed under various developmental conditions. Three genes (FgHyd1, FgHyd2, and FgHyd3) were constitutively expressed in all aerial structures examined (hyphae, conidia, and perithecia), and two genes (FgHyd1 and FgHyd2) were also expressed in submerged hyphae. FgHyd3 was exclusively expressed in aerial hyphae on solid surfaces, including rice grains. These genes showed markedly reduced expression in F. asiaticum, which was a closely related to F. graminearum but exhibited different mycological traits from F. graminearum. Phenotypic analyses of various gene deletion strains, including the quintuple deletion (ΔFgHyd12345) strain, confirmed that in addition to their typical functions, all five FgHyd genes were involved in other traits, such as conidiation, pathogenicity, and secondary metabolism in F. graminearum. Both RNA-seq and chemical analyses confirmed that ΔFgHyd led to overproduction of specific terpenoid compounds (e.g., trichothecenes), which has not been reported previously. Nevertheless, the lack of complete phenotypic loss of any of the traits examined, even in the ΔFgHyd12345 strain, and little cumulative action of all five FgHyd genes strongly suggest that all five hydrophobins are redundant in function and are not absolutely essential for these fungal traits in F. graminearum.

Fusarium asiaticum of the F. graminearum species complex causes head blight in small-grain cereals. The nivalenol (NIV) chemotypes of F. asiaticum is more common than the deoxynivalenol (DON) chemotypes of F. asiaticum or F. graminearum in Korea. To understand the prevalence of F. asiaticum–NIV in Korean cereals, we characterized the biological traits of 80 cereal isolates of F. asiaticum producing NIV or 3-acetyl-deoxynivalenol (3-ADON), and 54 F. graminearum with 3-ADON or 15-acetyl-deoxynivalenol (15-ADON). There was no significant difference in mycelial growth between the chemotypes, but F. asiaticum isolates grew approximately 30% faster than F. graminearum isolates on potato dextrose agar. Sexual and asexual reproduction capacities differed markedly between the two species. Both chemotypes of F. graminearum (3-ADON and 15-ADON) produced significantly higher numbers of perithecia and conidia than F. asiaticum–NIV. The highest level of mycotoxins (sum of trichothecenes and zearalenone) was produced by F. graminearum–3-ADON on rice medium, followed by F. graminearum–15-ADON, F. asiaticum–3-ADON, and F. asiaticum–NIV. Zearalenone levels were correlated with DON levels in some chemotypes, but not with NIV levels. Disease assessment on barley, maize, rice, and wheat revealed that both F. asiaticum and F. graminearum isolates were virulent toward all crops tested. However, there is a tendency that virulence levels of F. asiaticum–NIV isolates on rice were higher than those of F. graminearum isolates. Taken together, the phenotypic traits found among the Korean F. asiaticum–NIV isolates suggest an association with their host adaptation to certain environments in Korea.

Background To comprehensively understand the molecular mechanism of tamoxifen resistance (TamR) acquisition by epigenetically regulated genes, it is essential to identify pivotal genes by genome-wide methylation analysis and verify their function in xenograft animal model and cancer patients. Methods The MCF-7/TamR breast cancer cell line was developed and a genome-wide methylation array was performed. The methylation and expression of ELOVL2 was validated in cultured cells, xenografted tumor tissue, and breast cancer patients by methylation-specific PCR, qRT-PCR, Western blot analysis, and immunohistochemistry. Deregulation of ELOVL2 and THEM4 was achieved using siRNA or generating stable transfectants. Tam sensitivity, cell growth, and apoptosis were monitored by colorimetric and colony formation assay and flow cytometric analysis. Pathway analysis was performed to generate networks for the differentially methylated genes in the MCF-7/TamR cells and for the differentially expressed genes in the ELOVL2-overexpressing cells. Results Genome-wide methylation analysis in the MCF-7/TamR cells identified elongation of very-long chain fatty acid protein 2 (ELOVL2) to be significantly hypermethylated and downregulated, which was further verified in the tumor tissues from TamR breast cancer patients (n = 28) compared with those from Tam-sensitive (TamS) patients (n = 33) (P THEM4, which plays crucial roles in drug resistance. Conclusions ELOVL2 was hypermethylated and downregulated in TamR breast cancer patients compared with TamS patients. ELOVL2 is responsible for the recovery of Tam sensitivity. AKT- and ERα-hubbed networks are pivotal in ELOVL2 signaling, where THEM4 contributes to the relaying ELOVL2 signaling. This study implies that deregulation of a gene in fatty acid metabolism can lead to drug resistance, giving insight into the development of a new therapeutic strategy for drug-resistant breast cancer.

The depside and depsidone series compounds of polyketide origin accumulate in the cortical or medullary layers of lichen thalli. Despite the taxonomic and ecological significance of lichen chemistry and its pharmaceutical potentials, there has been no single piece of genetic evidence linking biosynthetic genes to lichen substances. Thus, we systematically analyzed lichen polyketide synthases (PKSs) for categorization and identification of the biosynthetic gene cluster (BGC) involved in depside/depsidone production. Our in-depth analysis of the interspecies PKS diversity in the genus Cladonia and a related Antarctic lichen, Stereocaulon alpinum, identified 45 BGC families, linking lichen PKSs to 15 previously characterized PKSs in nonlichenized fungi. Among these, we identified highly syntenic BGCs found exclusively in lichens producing atranorin (a depside). Heterologous expression of the putative atranorin PKS gene (coined atr1) yielded 4-O-demethylbarbatic acid, found in many lichens as a precursor compound, indicating an intermolecular cross-linking activity of Atr1 for depside formation. Subsequent introductions of tailoring enzymes into the heterologous host yielded atranorin, one of the most common cortical substances of macrolichens. Phylogenetic analysis of fungal PKS revealed that the Atr1 is in a novel PKS clade that included two conserved lichen-specific PKS families likely involved in biosynthesis of depsides and depsidones. Here, we provide a comprehensive catalog of PKS families of the genus Cladonia and functionally characterize a biosynthetic gene cluster from lichens, establishing a cornerstone for studying the genetics and chemical evolution of diverse lichen substances. IMPORTANCE Lichens play significant roles in ecosystem function and comprise about 20% of all known fungi. Polyketide-derived natural products accumulate in the cortical and medullary layers of lichen thalli, some of which play key roles in protection from biotic and abiotic stresses (e.g., herbivore attacks and UV irradiation). To date, however, no single lichen product has been linked to respective biosynthetic genes with genetic evidence. Here, we identified a gene cluster family responsible for biosynthesis of atranorin, a cortical substance found in diverse lichen species, by categorizing lichen polyketide synthase and reconstructing the atranorin biosynthetic pathway in a heterologous host. This study will help elucidate lichen secondary metabolism, harnessing the lichen's chemical diversity, hitherto obscured due to limited genetic information on lichens.

Lichens are prolific producers of natural products of polyketide origin. We previously described a culture of lichen-forming fungus (LFF) Cladonia macilenta that produces biruloquinone, a purple pigment that is a phenanthraquinone rarely found in nature. However, there was no genetic information on the biosynthesis of biruloquinone. To identify a biosynthetic gene cluster for biruloquinone, we mined polyketide synthase (PKS) genes from the genome sequence of a LFF isolated from thalli of C. macilenta. The 38 PKS in C. macilenta are highly diverse, many of which form phylogenetic clades with PKS previously characterized in non-lichenized fungi. We compared transcriptional profiles of the 38 PKS genes in two chemotypic variants, one producing biruloquinone and the other producing no appreciable metabolite in vitro. We identified a PKS gene (hereafter PKS21) that was highly upregulated in the LFF that produces biruloquinone. The boundaries of a putative biruloquinone gene cluster were demarcated by co-expression patterns of six clustered genes, including the PKS21. Biruloquinone gene clusters exhibited a high degree of synteny between related species. In this study we identified a novel PKS family responsible for the biosynthesis of biruloquinone through whole-transcriptome analysis.

An outbreak of new disease with leaf and stem blight symptom occurred at bracken-growing fields in Namhae-gun, Gyeongsangnam-do, Korea during the last 4 years. This new disease caused significant yield losses on bracken production in this area. We have collected diseased leaves and stems showing the blight symptom in May, July, and October 2018 to investigate causal pathogens. A total of 92 fungal isolates were obtained from the diseased samples and their pathogenicity was tested on healthy bracken leaves. From the total isolates, 22 isolates were able to produce the leaf blight symptom similar to the original one found in the fields. To identify two fungal pathogens which showed higher virulence levels compared to other pathogenic isolates, we constructed phylogenetic trees using the nucleotide sequences of genes for ribosomal RNA, RNA polymerase beta subunit, beta tubulin, and internal transcribed region. Most phylogenetic trees constructed indicate that both isolates, which are identical to each other, reside in a clade of the genus Didymella and possibly similar to D. rumicicola or D. acetosellae. Nevertheless, the exact identification of these pathogens at the species level needs further investigations. This is the first report of a blight disease on bracken by Didymella sp.

Lichens are prolific producers of natural products of polyketide origin. We previously described a culture of lichen-forming fungus (LFF) Cladonia macilenta that produces biruloquinone, a purple pigment that is a phenanthraquinone rarely found in nature. However, there was no genetic information on the biosynthesis of biruloquinone. To identify a biosynthetic gene cluster for biruloquinone, we mined polyketide synthase (PKS) genes from the genome sequence of a LFF isolated from thalli of C. macilenta. The 38 PKS in C. macilenta are highly diverse, many of which form phylogenetic clades with PKS previously characterized in non-lichenized fungi. We compared transcriptional profiles of the 38 PKS genes in two chemotypic variants, one producing biruloquinone and the other producing no appreciable metabolite in vitro. We identified a PKS gene (hereafter PKS21) that was highly upregulated in the LFF that produces biruloquinone. The boundaries of a putative biruloquinone gene cluster were demarcated by co-expression patterns of six clustered genes, including the PKS21. Biruloquinone gene clusters exhibited a high degree of synteny between related species. In this study we identified a novel PKS family responsible for the biosynthesis of biruloquinone through whole-transcriptome analysis.

Ginsenoside Rg3 exerts antiproliferation activity on cancer cells by regulating diverse noncoding RNAs. However, little is known about the role of long noncoding RNAs (lncRNAs) or their relationship with competitive endogenous RNA (ceRNA) in Rg3-treated cancer cells. Here, a lncRNA (ATXN8OS) was found to be downregulated via Rg3-mediated promoter hypermethylation in MCF-7 breast cancer cells. SiRNA-induced downregulation of ATXN8OS decreased cell proliferation but increased apoptosis, suggesting that the noncoding RNA possessed proproliferation activity. An in silico search for potential ATXN8OS-targeting microRNAs (miRs) identified a promising candidate (miR-424-5p) based on its high binding score. As expected, miR-424-5p suppressed proliferation and stimulated apoptosis of the MCF-7 cells. The in silico miR-target-gene prediction identified 200 potential target genes of miR-424-5p, which were subsequently narrowed down to seven that underwent hypermethylation at their promoter by Rg3. Among them, three genes (EYA1, DACH1, and CHRM3) were previously known oncogenes and were proven to be oppositely regulated by ATXN8OS and miR-424-5p. When taken together, Rg3 downregulated ATXN8OS that inhibited the tumor-suppressive miR-424-5p, leading to the downregulation of the oncogenic target genes.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the

Simple Summary Cold atmospheric plasma is being applied to treat cancer by virtue of its preferential anti-proliferative effect on cancer cells over normal cells. This study aimed to systemically determine the distribution of target genes regulated by the reactive oxygen species and non-reactive oxygen species constituents of the plasma. After analyzing genome-wide expression data for a leukemia and a melanoma cancer cell line from a public database followed by experimental approaches, PTGER3 and HSPA6 genes were found regulated by the non-reactive oxygen species and non-reactive nitrogen species constituents of the plasma in the cancer cells. This study could contribute to elucidate the molecular mechanism how each physicochemical constituent of the plasma induces the specific molecular changes in cancer cells. Abstract Cold atmospheric plasma (CAP) can induce cancer cell death. The majority of gene regulation studies have been biased towards reactive oxygen species (ROS) among the physicochemical components of CAP. The current study aimed to systemically determine the distribution of target genes regulated by the ROS and non-ROS constituents of CAP. Genome-wide expression data from a public database, which were obtained after treating U937 leukemia and SK-mel-147 melanoma cells with CAP or H2O2, were analyzed, and gene sets regulated by either or both of them were identified. The results showed 252 and 762 genes in H2O2-treated U937 and SK-mel-147 cells, respectively, and 112 and 843 genes in CAP-treated U937 and SK-mel-147 cells, respectively, with expression changes higher than two-fold. Notably, only four and two genes were regulated by H2O2 and CAP in common, respectively, indicating that non-ROS constituents were responsible for the regulation of the majority of CAP-regulated genes. Experiments using ROS and nitrogen oxide synthase (NOS) inhibitors demonstrated the ROS- and reactive nitrogen species (RNS)-independent regulation of PTGER3 and HSPA6 when U937 cancer cells were treated with CAP. Taken together, this study identified CAP-specific genes regulated by constituents other than ROS or RNS and could contribute to the annotation of the target genes of specific constituents in CAP.

Secondary metabolism is intimately linked to developmental processes in filamentous fungi. In a previous study, we revealed that several polyketide synthase (PKS) genes, including FgPKS7, are specifically induced during formation of the sexual fruiting body (perithecium) in the cereal pathogen Fusarium graminearum. The function of PKS7, which is essential for perithecial development and hyphal growth, is interchangeable between two phylogenetically related species, F. graminearum and F. asiaticum, but not conserved in the more distantly related species F. fujikuroi and F. neocosmosporiellum. FgPKS7 is under the control of global or upstream regulators including the mating-type (MAT) locus and regulates numerous downstream genes that are transcriptionally specific to and functionally essential for sexual development, several other PKS genes, and ABC transporter genes for azole resistance in F. graminearum. FgPKS7 is an essential element for proper sexual development and participates in a regulatory network controlled by the MAT locus. Although the chemical identity of FgPKS7 remains unclear, FgPKS7 is likely involved in chemical reaction(s) for synthesis of metabolite(s) that control or promote perithecial maturation in F. graminearum. This study provides in-depth insights into the direct role of secondary metabolites in sexual development of filamentous fungi.

To evaluate fire-induced damage to bridge structures, the thermal–structure interaction (TSI) fire analysis method is proposed, verified and applied to examine the behaviour of bridge superstructures with steel–concrete composite sections and prestressed concrete (PSC) exposed to fire loading. The proposed TSI fire analysis consists of two different modelling parts: thermal transfer analysis and thermodynamic structural analysis. The body temperature inside the structure is first calculated using fire curve boundary conditions in an overall non-linear transient thermal transfer analysis. Thermodynamic structural analysis is then performed based on the entire temperature and heat distribution in the structure. To validate the proposed method, comparisons are made with standard fire test results: the temperature distribution and the deflection of the steel–concrete composite superstructure agreed closely with the results of the standard fire test. The proposed fire analysis method is finally applied to two bridges with different superstructures (steel–concrete composite and PSC) damaged by recent fire events in Korea.

Paclitaxel (Tx) is a widely used therapeutic chemical for breast cancer treatment, however, cancer recurrence remains an obstacle for improved prognosis of cancer patients. In this study, cold atmospheric plasma (CAP) was tested for its potential to overcome the drug resistance. After developing Tx-resistant MCF-7 (MCF-7/TxR) breast cancer cells, CAP was applied to the cells, and its effect on the recovery of drug sensitivity was assessed in both cellular and molecular aspects. Sensitivity to Tx in the MCF-7/TxR cells was restored up to 73% by CAP. A comparison of genome-wide expression profiles between the TxR cells and the CAP-treated cells identified 49 genes that commonly appeared with significant changes. Notably, 20 genes, such as KIF13B, GOLM1, and TLE4, showed opposite expression profiles. The protein expression levels of selected genes, DAGLA and CEACAM1, were recovered to those of their parental cells by CAP. Taken together, CAP inhibited the growth of MCF-7/TxR cancer cells and recovered Tx sensitivity by resetting the expression of multiple drug resistance&ndash, related genes. These findings may contribute to extending the application of CAP to the treatment of TxR cancer.

RNA silencing pathways in filamentous fungi are composed of multiple component proteins and known to be involved in vegetative growth, virulence or sexual reproduction. We found that the tomato wilt fungus, Fusarium oxysporum f. sp. lycopersici (Fol), carries four homologues genes of Qde-2, an argonaute protein gene and one of the main component protein genes in Neurospora crassa. Gene targeting revealed that FoQde-2, one of the Qde-2 homologues in Fol, is involved in virulence to tomato but not in vegetative growth.

To assess the risk of fumonisin contamination in Korean cereals, we isolated colonies of the Fusarium fujikuroi species complex (FFSC) from barley, maize, rice and soybean samples from 2011 to 2015. A total of 878 FFSC strains were isolated mostly from maize and rice, and species identity of the isolates were determined using the DNA sequence of the translation elongation factor 1-α (TEF-1α) and RNA polymerase II (RPB2) genes. Fusaria recovered from Korean cereals included F. fujikuroi (317 isolates and a frequency of 36%), F. proliferatum (212 isolates and 24.1%), F. verticillioides (170 isolates and 19.4%), F. concentricum (86 strains and 9.8%), F. andiyazi (56 isolates and 6.4%), F. subglutinans (28 isolates and 3.2%), F. thapsinum (5 isolates and 0.6%), and F. circinatum (2 isolates and 0.2%). The rice samples were dominated by F. fujikuroi (47.4%), F. proliferatum (27.3%), and F. concentricum (15.1%), whereas maize samples were dominated by F. verticillioides (33.9%), F. fujikuroi (25.3%), and F. proliferatum (21.1%). A phylogenetic analysis of 70 representative isolates demonstrated that each species was resolved as genealogically exclusive in the ML tree. Fumonisin production potential was evaluated using a PCR assay for the fumonisin biosynthesis gene, FUM1 in all of the isolates. Most of the isolates tested (94%) were positive for FUM1. All of the isolates assigned to F. fujikuroi, F. proliferatum, F. verticillioides and F. thapsinum were positive for FUM1 irrespective of their host origin. Seventy-seven representative isolates positive for FUM1 were examined for fumonisin production in rice medium. The majority of F. proliferatum (26/27, 96.3%), F. verticillioides (16/17, 94.1%) and F. fujikuroi (19/25, 76.0%) produced both FB1 and FB2. Notably, 16 of 19 fumonisin-producing F. fujikuroi produced >1000μg/g of fumonisins (FB1+FB2) in rice medium, which is higher than that in previous reports. These results suggest that F. fujikuroi can produce high levels of fumonisins similar to F. verticillioides and F. proliferatum.

Fusarium asiaticum of the F. graminearum species complex causes head blight in small-grain cereals. The nivalenol (NIV) chemotypes of F. asiaticum is more common than the deoxynivalenol (DON) chemotypes of F. asiaticum or F. graminearum in Korea. To understand the prevalence of F. asiaticum–NIV in Korean cereals, we characterized the biological traits of 80 cereal isolates of F. asiaticum producing NIV or 3-acetyl-deoxynivalenol (3-ADON), and 54 F. graminearum with 3-ADON or 15-acetyl-deoxynivalenol (15-ADON). There was no significant difference in mycelial growth between the chemotypes, but F. asiaticum isolates grew approximately 30% faster than F. graminearum isolates on potato dextrose agar. Sexual and asexual reproduction capacities differed markedly between the two species. Both chemotypes of F. graminearum (3-ADON and 15-ADON) produced significantly higher numbers of perithecia and conidia than F. asiaticum–NIV. The highest level of mycotoxins (sum of trichothecenes and zearalenone) was produced by F. graminearum–3-ADON on rice medium, followed by F. graminearum–15-ADON, F. asiaticum–3-ADON, and F. asiaticum–NIV. Zearalenone levels were correlated with DON levels in some chemotypes, but not with NIV levels. Disease assessment on barley, maize, rice, and wheat revealed that both F. asiaticum and F. graminearum isolates were virulent toward all crops tested. However, there is a tendency that virulence levels of F. asiaticum–NIV isolates on rice were higher than those of F. graminearum isolates. Taken together, the phenotypic traits found among the Korean F. asiaticum–NIV isolates suggest an association with their host adaptation to certain environments in Korea.

Together with the Fusarium graminearum species complex, F. culmorum is a major member of the causal agents of Fusarium head blight on cereals such as wheat, barley and corn. It causes significant yield and quality losses and results in the contamination of grain with mycotoxins that are harmful to humans and animals. In Korea, F. culmorum is listed as a quarantine fungal species since it has yet to be found in the country. In this paper, we report that two isolates (J1 and J2) of F. culmorum were collected from the air at a rice paddy field in Korea. Species identification was confirmed by phylogenetic analysis using multi-locus sequence data derived from five genes encoding translation elongation factor, histone H3, phosphate permease, a reductase, and an ammonia ligase and by morphological comparison with reference strains. Both diagnostic PCR and chemical analysis confirmed that these F. culmorum isolates had the capacity to produce nivalenol, the trichothecene mycotoxin, in rice substrate. In addition, both isolates were pathogenic on wheat heads and corn stalks. This is the first report on the occurrence of F. culmorum in Korea.

The relationship between bone turnover markers and atherosclerosis is controversial.The purpose of this study was to determine the association of arterial stiffness with the levels of osteocalcin and C-terminal telopeptide of type I collagen (CTx).This cross-sectional study included 1691 men and 1913 women who participated in the medical examination programs of a hospital from March 2008 to December 2011.Arterial stiffness was estimated by brachial-ankle pulse wave velocity (baPWV). Osteocalcin and CTx were assayed by chemiluminescence immunoassay. Bone mineral density was measured by dual-energy X-ray absorptiometry.The mean baPWV was elevated at both ends of the osteocalcin quintiles in both men and women. However, the adjusted mean was higher in the lowest quintile of osteocalcin than in the other quintiles in men and women. Before adjustment, negative and positive relationships of baPWV with the levels of osteocalcin and CTx were observed in men (β = -0.123 and -0.078 for osteocalcin and CTx, respectively) and women (β = 0.151 and 0.193), respectively. After adjustment for age and metabolic parameters, osteocalcin was negatively related with baPWV at lower osteocalcin levels (Q1-Q2) in both sexes (in the fully adjusted model, β = -0.090 for men and -0.053 for women). No significant relationship was observed at higher values. The osteocalcin level was fit for a quadratic model for baPWV showing an inverse J-shape.The level of serum osteocalcin showed an inverse J-shaped relationship with arterial stiffness in both men and women. However, the association between the CTx level and arterial stiffness was not significant.

The study presented in this paper developed a sequential and universal bridge fire risk assessment procedure for constructed bridges, as well as countermeasures to reduce the risk level. Of interest were concrete and steel bridges on highways, except for certain special types of bridges. The fire risk assessment procedure consisted of three steps: ( a) preliminary risk analysis (PRA), which was a screening step to determine the likelihood of bridge fire events; ( b) simplified risk analysis (SRA), which was performed with the use of bridge inventory databases and checklists; and ( c) detailed risk analysis (DRA), which made use of computer simulation and collected data. A PRA determines whether a bridge requires an SRA on the basis of bridge clearance height and roadway conditions under the bridge. An SRA has three parts: analysis of fire occurrence, vulnerability to fire, and bridge importance. Itemized details were developed to evaluate the three parts and determine risk scoring. The SRA procedure identified which parts contributed to the resultant risk level. Reliability indexes concurrent with the latest ISO DP 10252 design code were used to determine the score of a given bridge and to perform an analysis to determine the risk level of 8,267 bridges in South Korea. The DRA was the final step, a detailed analysis of occurrence and vulnerability of a bridge mainly on the basis of computer simulation. This study provided guidelines to determine the DRA levels. Countermeasures also were developed for each of three risk levels (i.e., occurrence, vulnerability, and importance); the application of the countermeasures depended on the factors causing the risk.

The Southern corn leaf blight (SCLB) epidemic of 1970 devastated fields of T-cytoplasm corn planted in monoculture throughout the eastern United States. The epidemic was driven by race T, a previously unseen race of Cochliobolus heterostrophus. A second fungus, Phyllosticta zeae-maydis, with the same biological specificity, appeared coincidentally. Race T produces T-toxin, while Phyllosticta zeae-maydis produces PM-toxin, both host-selective polyketide toxins necessary for supervirulence. The present abundance of genome sequences offers an opportunity to tackle the evolutionary origins of T- and PM- toxin biosynthetic genes, previously thought unique to these species. Using the C. heterostrophus genes as probes, we identified orthologs in six additional Dothideomycete and three Eurotiomycete species. In stark contrast to the genetically fragmented race T Tox1 locus that encodes these genes, all newly found Tox1-like genes in other species reside at a single collinear locus. This compact arrangement, phylogenetic analyses, comparisons of Tox1 protein tree topology to a species tree, and Tox1 gene characteristics suggest that the locus is ancient and that some species, including C. heterostrophus, gained Tox1 by horizontal gene transfer. C. heterostrophus and Phyllosticta zeae-maydis did not exchange Tox1 DNA at the time of the SCLB epidemic, but how they acquired Tox1 remains uncertain. The presence of additional genes in Tox1-like clusters of other species, although not in C. heterostrophus and Phyllosticta zeae-maydis, suggests that the metabolites produced differ from T- and PM-toxin.

Fusarium graminearum, a member of the F. graminearum species complex, is a filamentous ascomycetous group that causes serious diseases in cereal crops. A screen of insertional mutants of F. graminearum, generated using a restriction enzyme-mediated integration method, identified a mutant designated R7048 showing pleiotropic phenotypes in several mycological traits. The vector insertion site in the R7048 genome was identified as the KpnI site within an ORF annotated as FGSG_06346 (designated FgVPS74), which showed similarity to vacuolar protein sorting-associated protein 74 in the baker yeast. Both targeted gene deletion and complementation analyses confirmed that FgVPS74 was involved in hyphal growth, conidiation, sexual development, mycotoxin production, and virulence towards host plants in F. graminearum. Electron microscopy analysis revealed no significant changes in morphology of the vacuole or other organelles, but a greater number of mitochondria were produced in the ΔFgVPS74 strain compared to the wild-type progenitor. Expression of a GFP-tagged FgVPS74 construct under its native promoter in the ΔFgVPS74 strain exhibited localization of GFP signal to putative vesicle structures, but not to the vacuolar membrane. Taken together, these findings demonstrated that a functional vacuolar protein-sorting pathway mediated by FgVPS74 is crucial for fungal growth and development in F. graminearum.

In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production.

The filamentous fungus Chromocrea spinulosa (Trichoderma spinulosum) exhibits both self-fertile (homothallic) and self-sterile (heterothallic) sexual reproductive behavior. Self-fertile strains produce progeny cohorts that are 50% homothallic, 50% heterothallic. Heterothallic progeny can mate only with homothallic strains, and progeny also segregate 50% homothallic, 50% heterothallic. Sequencing of the mating type (MAT) region of homothallic and heterothallic strains revealed that both carry an intact MAT1-1 locus with three MAT1-1 genes (MAT1-1-1, MAT1-1-2, MAT1-1-3), as previously described for the Sordariomycete group of filamentous fungi. Homothallic strains, however, have a second version of MAT with the MAT1-2 locus genetically linked to MAT1-1. In this version, the MAT1-1-1 open reading frame is split into a large and small fragment and the truncated ends are bordered by 115bp direct repeats (DR). The MAT1-2-1 gene and additional sequences are inserted between the repeats. To understand the mechanism whereby C. spinulosa can exhibit both homothallic and heterothallic behavior, we utilized molecular manipulation to delete one of the DRs from a homothallic strain and insert MAT1-2 into a heterothallic strain. Mating assays indicated that: i) the DRs are key to homothallic behavior, ii) looping out of MAT1-2-1 via intra-molecular homologous recombination between the DRs in self-fertile strains results in two nuclear types in an individual (one carrying both MAT1-1 and MAT1-2 and one carrying MAT1-1 only), iii) self-fertility is achieved by inter-nuclear recognition between these two nuclear types before meiosis, iv) the two types of nuclei are in unequal proportion, v) having both an intact MAT1-1-1 and MAT1-2-1 gene in a single nucleus is not sufficient for self-fertility, and vi) the large truncated MAT1-1-1 fragment is expressed. Comparisons with MAT regions of Trichoderma reesei and Trichoderma virens suggest that several crossovers between misaligned parental MAT chromosomes may have led to the MAT architecture of homothallic C. spinulosa., Author summary Fungi employ one of two mating tactics for sexual reproduction: self-sterile/heterothallic species can mate only with a genetically distinct partner while self-fertile/homothallic species do not require a partner. In ascomycetes, sexual reproduction is controlled by master regulators encoded by the mating-type (MAT) locus. The architecture of MAT differs in heterothallic versus homothallic species; heterothallics carry one of two forms (MAT1-1 or MAT1-2) per nucleus, whereas most homothallics carry both MAT forms in a single nucleus. There are intriguing exceptions. For example, the yeast models, Saccharomyces cerevisiae, and Schizosaccharomyces pombe undergo reversible MAT switching, not demonstrated in filamentous fungi. Here, we describe the mating mechanism in Chromocrea spinulosa (Trichoderma spinulosum), a filamentous ascomycete that exhibits both homothallic and heterothallic behavior. Self-fertile strains produce progeny cohorts that are 50% homothallic, 50% heterothallic. Self-sterile strains can mate only with homothallic strains, and when this occurs, homothallic and heterothallic progeny are also produced in a 1:1 ratio. By MAT sequencing and manipulation, we discovered unique MAT architecture and determined that self-fertility is achieved by deletion of MAT1-2 from most homothallic nuclei and subsequent inter-nuclear recognition between the resulting two, unevenly present, nuclear types in a common cytoplasm.

Fusarium fujikuroi causes bakanae (“foolish seedling”) disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles., Author summary Fusarium fujikuroi causes bakanae disease of rice. Infected seedlings appear to be taller and more slender when compared to healthy seedlings due to its ability to produce gibberellic acids (GAs). The disease is responsible for high yield losses, and its incidence varies with regions, rice cultivars grown and the aggressiveness of the fungal isolates. However, not all infected seedlings show bakanae symptoms: one of the isolates, B14, causes stunting and early withering of infected seedlings. The reason for the two pathotypes is not well understood. Researchers thought that the stunting phenotype was mostly caused by fungal-derived secondary metabolites such as fusaric acid, but there is no experimental evidence yet. B14 differs from the other strains by the presence of more PKS gene clusters, low expression of GA genes, lack of detectable levels of GAs and the production of high amounts of fumonisins in rice. Analysis of additional field isolates revealed a strong correlation between the pathotype (bakanae or stunting) and either GA or fumonisin production. Based on phylogenetic analyses, F. fujikuroi strains can be divided into two phylogenetically distinct subclades according to their pathotype and secondary metabolite profiles. This study provides new insights into the genomic variations and the population structure inside the species F. fujikuroi which will help to develop disease control strategies for this rice pathogen.

Fire event is the one of most severe event on the bridge. Fire event on the bridge has been reported annual average 8 cases in South Korea. Especially Bucheon viaduct fire cause catastrophic damage on the traffic system. This study developed and conducted fire risk assessment for existing bridge on the highway. Entire risk assessment procedure divided into 3 sections, PRA, SRA and DRA. PRA check fire scenario possibility. SRA check key parameter of fire risk by checklist. DRA is a precision risk assessment and find what part makes bridge high risk.

In recent decades, a combination of cytoreductive surgery and intraperitoneal chemotherapy has yielded improvements in the survival of patients with peritoneal carcinomatosis. Laparoscopic cytoreductive surgery and intraperitoneal chemotherapy comprise a challenging and rarely reported surgical procedure. Between November 2004 and February 2010, 29 patients underwent cytoreductive surgery and early postoperative intraperitoneal chemotherapy for peritoneal carcinomatosis secondary to colorectal cancer. Of the 29 patients, 15 underwent laparoscopic surgery and 14 underwent open surgery. The patient characteristics did not differ significantly between the two groups. Synchronous peritoneal carcinomatosis with a primary tumor was more common in the laparoscopic group, and the Gilly stage of peritoneal carcinomatosis was found more frequently in the open group. Complication rate and hospital stay were less in the laparoscopic group. However, the outcomes for the patients undergoing the combined treatment were similar between the two groups with respect to completeness of cytoreduction, operation morbidity, and overall survival. The laparoscopic group had a cytoreduction completeness of 86.7 % and an operative morbidity of 13.3 %. Operative mortality occurred for one patient after open surgery. Laparoscopic cytoreductive surgery and early postoperative intraperitoneal chemotherapy can be performed safely for selected patients with peritoneal carcinomatosis from colorectal cancer to a limited extent. Further studies with longer follow-up periods and larger numbers of patients are warranted to confirm the study findings.

Urinary and sexual dysfunction are recognized complications of rectal cancer surgery in men. This study compared robot-assisted total mesorectal excision (RTME) and laparoscopic total mesorectal excision (LTME) with regard to these functional outcomes. A series of 32 men who underwent RTME between February 1, 2009 and December 31, 2010 were matched 1:1 with patients who underwent LTME. The matching criteria were age, body mass index, tumor distance from the anal verge, neoadjuvant chemoradiation therapy, and tumor stage. Urinary and erectile function were evaluated using the International Prostatic Symptom Score (IPSS) and the five-item version of the International Index of Erectile Function (IIEF-5) scale. Data were collected from the two groups at baseline and at 3, 6, and 12 months after surgery and compared. The mean IPSS score did not differ between the two groups at baseline at any point of measurement. The mean baseline IIEF-5 score was similar between the two groups and was decreased at 3 months. The mean IIEF-5 score was significantly higher in the RTME group at 6 months than in the LTME group (14.1 ± 6.1 vs. 9.4 ± 6.6; p = 0.024). The interval decrease in IIEF-5 scores was significantly higher in the LTME group than in the RTME group at 6 months (4.9 ± 4.5 vs. 9.2 ± 4.7; p = 0.030). The men in the RTME group experienced earlier restoration of erectile function than did those in the LTME group. Bladder function was similar during the 12 months after RTME or LTME.

The blast damage behaviors for concrete panels retrofitted with steel plates exposed to blast loading are investigated. In order to enhance the reliability of the simulation results, the equation of state, strength, and failure model of materials are implemented in an explicit analysis program, AUTODYN. In particular, the implemented formulation includes the rate-dependent plasticity and damage softening; the non-linear strain and strain-rate hardening and non-linear strain softening. Furthermore, simplified and idealized 2D axis-symmetry, 3D, and parallel 3D simulations are compared in order to achieve accurate and efficient computation running times using multi-solver coupling method. Comparing the 2D axis-symmetry and 3D models, 2D model is stiffer and has a smaller deflection than the 3D models by geometries of retrofit material. The result of 3D numerical simulation becomes mesh size dependent, because of the explosive characteristics and mechanical properties of concrete. The parallel 3D simulation shows good scalability up to 15 processors and can be simulated at very high speed-up, while still consuming a reasonable amount of run times. Also, the retrofitted concrete panels are compared to the non-retrofitted concrete panel; maximum deflection and maximum deflection ratio are reduced by 1 mm, 3 mm, and 5 mm thick steel plates, scabbing can be prevented by retrofitted with steel plates. The simulation result shows good agreement with the experimental result. Finally, discussion on the numerical results with respect to code criteria and damage localization in retrofitted concrete panels is presented.

The objective of this research is to evaluate of global and local damage for steel-concrete composite structures under highway bridge exposed to fire loading. To enhance the accuracy and efficiency of the numerical analysis, the proposed transient nonlinear thermal structure interaction(TSI) parallel fire analysis method is implemented in ANSYS. To validate the TSI parallel fire analysis method, a comparison is made with the standard fire test results. The proposed TSI parallel fire analysis method is applied to fire damage analysis and performance evaluation for Buchen highway bridge. The result of analysis, temperature of low flange and web are exceed the critical temperature. The deflection and deformation state show good agreement with the fire accident of buchen highway bridge.

The blast damage behaviors of steel-plated and glass fiber reinforced polymer (GFRP)-plated concrete panels exposed to explosive are investigated. In order to improve efficiency and accuracy of numerical analysis, the multi-physics method of coupled Eulerian-Lagrangian discretization is used. To enhance the reliability of the simulation results, the equation of state, strength, and failure model of materials are implemented in an explicit program, AUTODYN. In particular, the implemented formulation includes the strain rate-dependent plasticity for concrete and a transversely isotropic elastic constitutive model for GFRP. The retrofitted concrete panels are compared to non-retrofitted concrete panels, and the deflection and deflection ratio are reduced by steel and GFRP plates. To validate the implemented material models and analysis method, a comparison is made with the reported experimental results. The maximum deflection of the panel from the numerical analysis agrees closely with the results of the experiments. Finally, a discussion of the numerical results with respect to code criteria and energy absorption capacity is presented.

Microbial Safety Team, National Academy of Agricultural Science, RDA, Suwon 441-707, Korea(Received on September 1, 2012; Revised on September 20, 2012; Accepted on September 23, 2012)Several species belonging to the Gibberella fujikuroispecies ( Gf ) complex are commonly associated with riceand corn, not only causing serious diseases, but alsoproducing fumonisins, a group of mycotoxins harmfulto animals and humans. To characterize the populationstructure of the putative fumonisin-producing Gf complexin Korea, we obtained 276 candidate isolates from riceand corn harvested in 2009 and 2010 by diagnosticpolymerase chain reaction with several specific primersets. Phylogenetic trees were constructed using multilocussequences (combined RPB2 and EF1A, totaling 1.6 kb)from these isolates. Among the 135 isolates from rice,F. fujikuroi (teleomorph: G. fujikuroi; 59.3%) and F.proliferatum (G. intermedia; 13.3%) were predominant,followed by F. concentricum (5.9%). Additionally, twenty-five (18.5%) rice isolates belonged in a distinct sub-clade of F. commune, a non-member of the Gf complex.In contrast, F. verticillioides was the most predominantspecies (38.3%) among the 141 corn isolates, and followedby F. fujikuroi (27.7%), F. proliferatum (14.9%), F. sub-glutinans (7.1%), and F. concentricum (2.8%). A singlemating type ( MAT1-1) was found predominantly amongthe Gf complex isolates examined. Possible distinct sub-clades were detected within the populations of F. fujikuroiand F. proliferatum; however, this needs further confir-mation. This is the first reported population-level charac-terization of putative fumonisin-producing Gf complexassociated with rice and corn in Korea.Keywords : fumonisin, Gibberella fujikuroi species com-plex, mating-type distribution, population structure, speciescompositionThe genus Fusarium is one of the most important fungalisolates associated with rice and corn because some membersof this group, such as the F. graminearum species complexand Gibberella fujikuroi species complex, are mycotoxinproducers as well as plant pathogens. The G. fujikuroi speciescomplex, a monophyletic lineage, consists of at least ninebiological species, formerly designated as mating populations(MPs) (Britz et al., 1999; Leslie 1999; Leslie et al., 2004),and numerous additional Fusarium anamorphs that areassigned to phylogenetically distinct species (Nirenbergand O'Donnell 1998; O'Donnell et al., 1998; O'Donnell etal., 2000). The Fusarium species in the G. fujikuroi species complexare distributed worldwide and cause serious diseases in awide variety of agricultural crops (Leslie 1999). These speciesalso produce a variety of toxic secondary metabolites andmycotoxins, including gibberellic acid (Cerda-Olmedo etal., 1994), moniliformin (Marasas et al., 1986), and fumoni-sins (Rheeder et al., 2002). Among these, fumonisins, agroup of polyketide-derived mycotoxins, have gained con-siderable attention because they have been associated withesophageal and liver cancer (Desjardins, 2006).To date, at least 15 Fusarium species, most belonging tothe G. fujikuroi species complex, are known to producefumonisins using a biochemical pathway catalyzed by apolyketide synthase (FUM1) (Proctor et al., 1999). Only afew species outside the G. fujikuroi species complex, suchas F. oxysporum, have the capacity for fumonisin produc-tion (Rheeder et al., 2002).As both rice grains and corn kernels are capable ofharboring the toxigenic G. fujikuroi species complex duringthe growing season, monitoring the fumonisin-producingFusarium species on these substrates is important. Severalspecies within the G. fujikuroi species complex are knownto be frequently associated with rice and corn. Three fumonisin-producing species, F. fujikuroi (MP-C,teleomorph: G. fujikuroi), F. proliferatum (MP-D, G. inter-media), and F. verticillioides (MP-A, G. moniliformis ), havebeen isolated from rice plants showing bakanae symptomsin Asia, Africa, Europe, and the United States (Amatulli etal., 2010; Carter et al., 2008; Desjardins et al., 2000;Desjardins et al., 1997; Hsuan et al., 2011; Wulff et al.,2010; Zainudin et al., 2008). The incidence of the latter wasrelatively lower than the first two species. F. verticillioidesis the most prevalent species on corn worldwide (Chulze et

Gibberellea fujikuroi species (Gf) complex comprises at least 15 species, most of which not only causes seriousplant diseases, but also produces mycotoxins including fumonisins. Here, we focused on the abilities of thefield isolates belonging to the Gf complex associated with rice and corn, respectively in Korea to producefumonisin, all of which were confirmed to carry FUM1, the polyketide synthase gene essential for fumonisinbiosynthesis. A total of 88 Gf complex isolates (55 F. fujikuroi, 10 F. verticillioides, 20 F. proliferatum, 2 F.subglutinans, and 1 F. concentricum), and 4 isolates of F. commune, which is a non-member of Gf complex,were grown on rice substrate and determined for their production levels of fumonisins by a HPLC method.Most isolates of F. verticillioides and F. proliferatum, regardless of host origins, produced fumonisin B1 and B2at diverse ranges of levels (0.5−2,686.4 μg/g, and 0.7−1,497.6 μg/g, respectively). In contrast, all the isolates ofF. fujikuroi and other Fusarium species examined produced no fumonisins or only trace amounts (

Department of Medical Biotechnology, Soonchunhyang University, Asan 336-745, Korea(Received on May 12, 2012; Revised on May 23, 2012; Accepted on May 23, 2012)Protein phosphatase 2A (PP2A), a family of serine/threonine protein phosphatases, plays an important rolein balancing the phosphorylation status of cellularproteins for regulating diverse biological functions ineukaryotic organisms. Despite intensive studies inmammals, limited information on its role is available infilamentous fungi. Here, we investigated the functionalroles of genes for a putative B' delta regulatory subunit(FgPP2AR) and a catalytic subunit ( FgPP2AC) of PP2Ain a filamentous ascomycete, Fusarium graminearum.Molecular characterization of an insertional mutant ofthis plant pathogenic fungus allowed us to identify theroles of FgPP2AR. Targeted gene replacement and com-plementation analyses demonstrated that the deletion ofFgPP2AR, which was constitutively expressed in allgrowth stages, caused drastic changes in hyphal growth,conidia morphology/germination, gene expression formycotoxin production, sexual development and patho-genicity. In particular, overproduction of aberrantcylindrical-shaped conidia is suggestive of arthroconi-dial induction in the ΔFgPP2AR strain, which has neverbeen described in F. graminearum. In contrast, theΔFgPP2AC strain was not significantly different fromits wild-type progenitor in conidiation, trichothecenegene expression, and pathogenicity; however, it showedreduced hyphal growth and no perithecial formation.The double-deletion ΔFgPP2AR;ΔFgPP2AC strain hadmore severe defects than single-deletion strains in allexamined phenotypes. Taken together, our results indi-cate that both the putative regulatory and catalytic sub-units of PP2A are involved in various cellular processesfor fungal development in F. graminearum. Keywords : aberrant conidia morphology, B regulatorysubunit, catalytic subunit, Fusarium graminearum, proteinphosphatase 2A Molecular regulation of many biological functions ineukaryotic organisms is mediated by protein posttranslationalmodifications. Protein phosphorylation is one of the majormechanisms in this regulatory system. In particular, rever-sible phosphorylation of cellular proteins in response toexternal or intracellular stimuli is an essential step for signaltransmission to specific transcription factors that regulatedownstream genes in a specific signal transduction cascade(Liu et al., 2000; Luan, 2003). The phosphorylation statusof target proteins is modulated by the reversible activities ofprotein kinases and protein phosphatases. Serine/threonineprotein phosphatases (PPs), which catalyze the dephos-phorylation of proteins, comprise two different structuralfamilies based on biochemical characteristics: type 1 (PP1)and type 2 (PP2). PP2s are subsequently divided into threegroups (2A, 2B, and 2C). Protein phosphatase 2A (PP2A)holoenzyme, a member of the PP2 family, consists of a coredimer comprising a catalytic C subunit (PP2AC) and ascaffolding A subunit, which can be complexed with a thirdvariable regulatory B subunit (PP2AB) to form a hetero-trimer (Mumby, 2007). The variable B subunits, which candetermine the substrate specificity, catalytic activity, andcellular localization of PP2A, are grouped into four un-related families, namely B (PR55), B' (B56/PR61), B''(PR48/59/72/130) and B''' (PR93/110) based on structuralsimilarities. Among the B regulatory subunits, the B' sub-unit is the most diverse, consisting of five isoforms (alpha,beta, gamma, delta, and epsilon) (McCright et al., 1996;Muneer et al., 2002; Sontag, 2001). The diversity of possiblecombinations of these subunits allows the PP2A holo-enzyme to play roles in the regulation of a wide range ofcellular and metabolic processes in a variety of organisms(Gallego and Virshup, 2005; Lechward et al., 2001). Despiteintensive investigations in human and model organisms,limited information on the role of PP2A is available infilamentous fungi.Fusarium graminearum (teleomorph: Gibberella zeae) isa filamentous ascomycetous fungus that threatens bothplant and animal health not only by causing disease but also

The velvet genes are conserved in ascomycetous fungi and function as global regulators of differentiation and secondary metabolism. Here, we characterized one of the velvet genes, designated FgVelB, in the plant-pathogenic fungus Fusarium graminearum, which causes fusarium head blight in cereals and produces mycotoxins within plants. FgVelB-deleted (ΔFgVelB) strains produced fewer aerial mycelia with less pigmentation than those of the wild-type (WT) during vegetative growth. Under sexual development conditions, the ΔFgVelB strains produced no fruiting bodies but retained male fertility, and conidiation was threefold higher compared with the WT strain. Production of trichothecene and zearalenone was dramatically reduced compared with the WT strain. In addition, the ΔFgVelB strains were incapable of colonizing host plant tissues. Transcript analyses revealed that FgVelB was highly expressed during the sexual development stage, and may be regulated by a mitogen-activated protein kinase cascade. Microarray analysis showed that FgVelB affects regulatory pathways mediated by the mating-type loci and a G-protein alpha subunit, as well as primary and secondary metabolism. These results suggest that FgVelB has diverse biological functions, probably by acting as a member of a possible velvet protein complex, although identification of the FgVelB-FgVeA complex and the determination of its roles require further investigation.

Fusarium verticillioides (teleomorph: Gibberella moniliformis), a member of the Gibberellea fujikuroi species complex, causes rots of corn stalks and ears, and produces a group of mycotoxins known as fumonisins that are harmful to animals and humans. Here, we focus on the development of a species-specific PCR primer set for differentiating F. verticillioides from other fumonisin-producing Fusarium species belonging to the species complex, such as F. proliferatum, F. fujikuroi, and F. subglutinans that are frequently associated with corn. The specific primers (RVERT1 and RVERT2) derived from the nucleotide sequences of RNA polymerase II beta subunit (RPB2) gene amplified a 208 bp-DNA fragment from only F. verticillioides isolates among the potential fumonisin-producing species examined; all of these isolates were shown to carry FUM1 required for fumonisin biosynthesis. The PCR detection limit using this specific primer set was approximately 0.125 pg/ genomic DNA of F. verticillioides. In addition, the F. verticillioides-specfic fragment was successfully amplified from genomic DNAs of corn samples contaminated with Fusarium spp. This primer set would provide a useful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in cereal samples.

Department of Medical Biotehcnology, Soonchunhyang University, Asan 336-745, Korea(Received on October 25, 2011; Accepted on November 2, 2011)The filamentous fungus Fusarium graminearum is animportant cereal pathogen. Although quantitative real-time PCR (qRT-PCR) is commonly used to analyze theexpression of important fungal genes, no detailedvalidation of reference genes for the normalization ofqRT-PCR data has been performed in this fungus.Here, we evaluated 15 candidate genes as references,including those previously described as housekeepinggenes and those selected from the whole transcriptomesequencing data. By a combination of three statisticalalgorithms (BestKeeper, geNorm, and NormFinder),the variation in the expression of these genes wasassessed under different culture conditions that favoredmycelial growth, sexual development, and trichothecenemycotoxin production. When favoring mycelial growth,GzFLO and GzUBH expression were most stable incomplete medium. Both EF1A and GzRPS16 expressionwere relatively stable under all conditions on carrotagar, including mycelial growth and the subsequentperithecial induction stage. These two genes were alsomost stable during trichothecene production. For thecombined data set, GzUBH and EF1A were selected asthe most stable. Thus, these genes are suitable referencegenes for accurate normalization of qRT-PCR data forgene expression analyses of F. graminearum and otherrelated fungi. Keywords : Fusarium graminearum, gene expression,quantitative real-time PCR, reference genesQuantitative real-time PCR (qRT-PCR) is the most sensi-tive and specific method for quantifying mRNA expressionlevels of individual target genes of interest. qRT-PCRexpression analysis has several advantages over otherconventional methods, but requires data normalization withappropriate reference gene(s) whose expression should beunaffected throughout the study conditions (Anderson etal., 2004; Bustin et al., 2004; Gutierrez et al., 2008). Severalhousekeeping genes involved in basic cellular functions,such as ribosomal genes, and those encoding actin, beta-tubulin (BTUB), translation elongation factor (EF1), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) havebeen used as reference genes for qRT-PCR in humans andother model organisms. However, several studies havedemonstrated that some of these traditional housekeepinggenes are unsuitable references because their expressionlevels vary under certain conditions (Czechowski et al.,2005; Dheda et al., 2004; Lee et al., 2002; Suzuki et al.,2000). Therefore, it is necessary to evaluate candidatereference genes for qRT-PCR expression analysis underspecific experimental conditions in the organism to bestudied. The filamentous fungus Fusarium graminearum (telo-morph: Gibberella zeae) is an important cereal pathogen,and produces mycotoxins that are harmful to humans andanimals (Desjardins and Proctor, 2007; McMullen et al.,1997). Complete genome sequencing of F. graminearum(Cuomo et al., 2007) has allowed for genome-wide expre-ssion profiling using DNA microarrays under variousdevelopmental and physiological conditions, such as my-celial growth (Guldener et al., 2006) sexual development(Hallen et al., 2007), germination (Seong et al., 2008) andpathogenesis (Guldener et al., 2006; Stephens et al., 2008).Although the microarray data in these studies were notvalidated by qRT-PCR, it has become the most commonmethod to obtain specific expression patterns of genesidentified from gene expression profiles (Chen et al., 2011;Liu et al., 2011; Pandolfi et al., 2010) or functional studies(Lee et al., 2009; Lysoe et al., 2009). However, theexpression stabilities of the housekeeping genes used asreferences for qRT-PCR analysis in even these studies havenot been properly validated. The objective of this study was to identify F. gramine-arum reference genes suitable for normalization of qRT-PCR data under different conditions such as mycelialgrowth, sexual development, and mycotoxin production.We selected 15 candidate references, including the commonhousekeeping genes used in previous studies (Chen et al.,2011; Lee et al., 2009; Liu et al., 2011; Lysoe et al., 2009;Pandolfi et al., 2010) and a new set of genes selected from

Fusarium verticillioides (teleomorph: Gibberella moniliformis), a member of the Gibberellea fujikuroi speciescomplex, causes rots of corn stalks and ears, and produces a group of mycotoxins known as fumonisins thatare harmful to animals and humans. Here, we focus on the development of a species-specific PCR primer setfor differentiating F. verticillioides from other fumonisin-producing Fusarium species belonging to the speciescomplex, such as F. proliferatum, F. fujikuroi, and F. subglutinans that are frequently associated with corn.The specific primers (RVERT1 and RVERT2) derived from the nucleotide sequences of RNA polymerase IIbeta subunit (RPB2) gene amplified a 208 bp-DNA fragment from only F. verticillioides isolates among thepotential fumonisin-producing species examined; all of these isolates were shown to carry FUM1 required forfumonisin biosynthesis. The PCR detection limit using this specific primer set was approximately 0.125 pg/μlgenomic DNA of F. verticillioides. In addition, the F. verticillioides-specfic fragment was successfully amplifiedfrom genomic DNAs of corn samples contaminated with Fusarium spp. This primer set would provide auseful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains incereal samples.

Xylogone ganodermophthora, an ascomycetous fungus, is known to cause yellow rot in the cultivated mushroom Ganoderma lucidum. In this study, we investigated the dissemination of this fungal pathogen in G. lucidum grown in cultivation houses. To determine the role of ascospores produced by X. ganodermophthora in disease development, we constructed a green fluorescent protein-labeled transgenic strain. This X. ganodermophthora strain produced a number of ascomata in the tissues of oak logs on which G. lucidum had been grown and on the mushroom fruit bodies. However, the ascospores released from the ascomata were not able to germinate on water agar or potato dextrose agar. Moreover, less than 0.1% of the ascospores showed green fluorescence, indicating that most ascospores of X. ganodermophthora were not viable. To determine the manner in which X. ganodermophthora disseminates, diseased oak logs were either buried in isolated soil beds as soil-borne inocula or placed around soil beds as air-borne inocula. In addition, culture bottles in which G. lucidum mycelia had been grown were placed on each floor of a five-floor shelf near X. ganodermophthora inocula. One year after cultivation, yellow rot occurred in almost all of the oak logs in the soil beds, including those in beds without soil-borne inocula. In contrast, none of the G. lucidum in the culture bottles was infected, suggesting that dissemination of X. ganodermophthora can occur via the cultivation soil.

Gibberella zeae (anamorph: Fusarium graminearum), a homothallic (self-ferile) ascomycete with ubiquitous geographic distribution, causes serious diseases in several cereal crops. Ascospores (sexual spores) produced by this fungal pathogen have been suggested as the main source of primary inoculum in disease development. Here, we report the function of a gene designated GzRUM1, which is essential for ascospore formation in G. zeae. The deduced product of GzRUM1 showed significant similarities to the human retinoblastoma (tumor suppressor) binding protein 2 and a transcriptional repressor, Rum1 in the corn smut fungus (Ustilago maydis). The transcript of GzRUM1 was detected during the both vegetative and sexual stages, but was more highly accumulated during the latter stage. In addition, no GzRUM1 transcript was detected in a G. zeae strain lacking a mating-type gene (MAT1-2), a master regulator for sexual development in G. zeae. Targeted deletion of GzRUM1 caused no dramatic changes in several traits except ascospore formation. The GzRUM1 strain produced perithecia (sexual fruit bodies) but not asci nor ascospores within them. This specific defect leading to an arrest in ascospore development suggests that GzRUM1, as Rum1 in U. maydis, functions as a transcriptional regulator during sexual reproduction in G. zeae.

Fusarium species, a large group of plant pathogens, potentially pose quarantine concerns worldwide. Here, we focus on the development of a method for detecting four Fusarium species in quarantined plants in Korea: F. solani f. sp. cucurbitae, F. stilboides, F. redolens, and F. semitectum var. majus. Species-specific primers were designed from the nucleotide sequences of either the translation elongation factor-1 alpha (TEF1) gene or RNA polymerase II subunit (RPB2) gene. Two different primer sets derived from TEF1, all specific to F. solani f. sp. cucurbitae, were able to differentiate the two races (1 and 2) of this species. A set of nested primers for each race was designed to confirm the PCR results. Similarly, two primer sets derived from RPB2 successfully amplified specific fragments from five F. stilboides isolates grouped within a single phylogenetic clade. A specific TEF1 primer set amplified a DNA fragment from only four of the 12 F. redolens strains examined, which were grouped within a single phylogenetic clade. All of the F. semitectum var. majus isolates could be specifically detected with a single RPB2 primer set. The specificity of the primer sets developed here was confirmed using a total of 130 Fusarium isolates.

Since existing constitutive models developed for confined normal strength concrete overestimate ductility when they are applied to confined high strength concrete, these models cannot be directly applied to confined high strength concrete. In an effort to solve this problem, an accurate stress-strain relationship of the hihg strength concrete needs to be formulated by examining the confinement effects due to increase of the concrete strength. In this study, a constitutive model is developed to express the stress-strain relationship of confined high strength concrete by carrying out regression analysis of the main parameters affection strength and ductile behavior of reinforced high strength concrete columns. Twenty-five test specimens were chosen from the reported experimental studies in the literature. The experimental results of stress-strain relationships of show a good agreement with results of the stress-strain relationships of suggested high strength concrete, covering a strength range between 60 and 124 MPa.

The performance evaluation of reinforcement concrete structure is carried out as a function of the following performance influencing factors: (1) the strength of concrete, (2) longitudinal reinforcement, (3) transverse reinforcement, (4) aspect ratio, and (5) axial force. With various values of the five parameters, eigenvalue analysis and non-linear static analysis were performed to investigate the structural yield displacement, yield basis shear force, and static performance of ductility ratio. In addition, the performance evaluation is carried out according to the modified capacity spectrum method (FEMA-440) using the results of non-linear static analysis, and the effect of each parameter on performance point is analyzed. Based on the result of eigenvalue analysis and non-linear static analysis indicates, that the natural period and the ductility ratio are affected more by the structural properties than the material properties. In case of the analysis of the criterion of performance points, the effect of section shape is one of the important factors together with natural period and ductility ratio.

We identified two syntaxin-like SNARE genes, named GzSYN1 and GzSYN2, from the plant pathogenic ascomycete Gibberella zeae, and characterized the functions and cellular localization of these genes. The GzSYN1 deletion mutant (Deltagzsyn1) had 71% reduced hyphal growth compared to the wild-type strain, but produced perithecia with normal ascospores. Deltagzsyn2 had the same hyphal growth rate as the wild-type, but completely lost both self and female fertility. When Deltagzsyn2 was spermatized for Deltamat1-1 or Deltamat1-2 strains, it retained its male fertility, but the ascus shape was abnormal and ascospore delimitation was delayed. The Deltagzsyn1 and Deltagzsyn2 virulence on barley was reduced by 67% and 75%, respectively, compared to the wild-type. The GFP::GzSYN1 fusion protein was localized in vesicles, vacuoles, plasma membranes, and septa, whereas GFP::GzSYN2 was found only in plasma membranes and septa. These results suggest that syntaxins have key roles in fungal development and virulence in G. zeae.

Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.