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Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain’s genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

Coral−algae symbiosis represents the trophic and structural basis of coral reef ecosystems. However, despite global threats to coral reefs and the dependence of coral health and stress resistance upon such mutualisms, little is known about the community ecology of endosymbiotic Symbiodiniaceae. Concepts and methods from metacommunity ecology may be used to help us understand the assembly and stability of symbiont communities and the mutualisms they comprise. In this study, we sampled colonies of the symbiont-generalist coral Galaxea fascicularis in southwestern Japan and assessed the effects of environmental and host factors on Symbiodinia ceae community composition, while simultaneously exploring residual correlations among symbiont types that may reflect non-random assembly processes such as species interactions. We metabarcoded the Symbiodiniaceae ribosomal internal transcribed spacer 2 (ITS2) region and characterized the endosymbiotic community using 2 different OTU identity cut-offs, and analyzed them with generalized dissimilarity modeling and joint species distribution modeling. We found that Symbiodiniaceae form discrete communities characterized by the dominance of ITS2 types C1, C21a, or D1, that are each associated with a different suite of co-occurring background types and tend to exclude each other in an endosymbiotic community. The communities showed modest responses to temperature, water depth, host genotype, polyp size, and bleaching status, and there was local sequence variation within the ITS2 types. After accounting for the effects of those variables, residual correlations remained in community composition, pointing to the possibility that Symbiodiniaceae community assembly in corals may be structured by interspecific competitive or facilitating interactions rather than only exogenous variables.

This research aimed to explore the anticancer potential in the protein and hydrolysate protein produced by epiphytic bacteria associated with marine algae on the in vitro model of lung cancer LK-2. Protein was isolated from the bacterium Enterobacter unhas strain SG-A1 in which has an association with brown algae, Sargassum sp. A fractionation method has used in separating the protein. It was used as a dialysis process added by Tris HCl to conduct pre-purified of the fractions, and the pepsin enzyme was used in producing protein hydrolysate (pH 2.0, 37 C) at 3:100 ratio of enzyme-substrate. Pre-screening of toxicity can be conducted using BSLT or Brine Shrimp Lethality Test. The cytotoxic effects of protein and protein hydrolysate on LK-2 cell lines during 16 h were determined by the yellow tetrazolium salt 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The BSLT test revealed that the hydrolysate fractions were very toxic. It showed a higher magnitude of decreasing (significance p < 0.05) in cell viability in LK-2 cells. These findings suggested that the hydrolysate of protein produced by epiphytic bacteria brown algae associated, Sargassum sp. has a high probability of being used as cancer therapy agents.

MicroRNAs can regulate inflammatory responses by modulating macrophage polarization. Although microRNA miR-21 is linked to crucial processes involved in inflammatory responses, its precise role in macrophage polarization is controversial. In this study, we investigated the functional relevance of endogenous miRNA-21 and the role of exosomes. RAW 264.7 macrophages were transfected with miR-21 plasmid, and the inflammatory response was evaluated by flow cytometry, phagocytosis, and real-time PCR analysis of inflammatory cytokines. To understand the signaling pathways' role, the cells were treated with inhibitors specific for PI3K or NFĸB. Exosomes from transfected cells were used to study the paracrine action of miR-21 on naive macrophages. Overexpression of miR-21 resulted in significant upregulation of pro-inflammatory cytokines, pushing the cells towards a pro-inflammatory phenotype, with partial involvement of PI3K and NFĸB signal pathways. The cells also secreted miR-21 rich exosomes, which, on delivery to naive macrophages, caused them to exhibit pro-inflammatory activity. The presence of miR-21 inhibitor quenched the inflammatory response. This study validates the pro-inflammatory property of miR-21 with a tendency to foster an inflammatory milieu. Our findings also reinforce the dual importance of exosomal miR-21 as a biomarker and therapeutic target in inflammatory conditions.

Fusarium graminearum produces trichothecene mycotoxins in infected grains and axenic liquid culture. A proposed regulatory model of trichothecene biosynthesis was examined in relation to nitrogen utilization. First, we showed that an important factor for the stimulation of trichothecene biosynthesis was not the occurrence of agmatine as a specific inducer molecule, but rather continuous acidification of the liquid culture medium arising from agmatine catabolism. When the pH of the L-Gln synthetic medium was frequently adjusted to the pH of the agmatine culture, trichothecene productivity of the L-Gln culture was equal to that of the agmatine culture. For efficient trichothecene biosynthesis, the culture pH should be lowered at an appropriate time point during the early growth stage. Second, we re-evaluated the role of the nitrogen regulatory GATA transcription factor AreA in trichothecene biosynthesis. Since Tri6 encodes a transcription factor indispensable for trichothecene biosynthesis, all fifteen AreA-binding consensus sequences in the Tri6 promoter were mutated. The mutant could catabolize L-Phe as the sole nitrogen source; furthermore, the pH profile of the synthetic L-Phe medium (initial pH 4.2) was the same as that of the wild-type (WT) strain. Under such conditions, the promoter mutant exhibited approximately 72% of the trichothecene productivity compared to the WT strain. Thus, F. graminearum AreA (FgAreAp) is dispensable for the functioning of the Tri6 promoter, but it contributes to the increased production of mycotoxin under mildly acidic conditions to some extent. Further investigations on the culture pH revealed that extremely low pH bypasses the function of FgAreAp.

While Fusarium graminearum readily produces trichothecenes in complex media containing sucrose as the carbon source (YS_60), the amount of the mycotoxin is quite limited when other sugars, such as glucose and fructose, are used. We found that autoclaving of media containing fructose and yeast extract (YF_60) results in the formation of inhibitors of trichothecene biosynthesis by F. graminearum JCM 9873, a strain that produces 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Removal of the solvent fraction from the autoclaved media after ethyl acetate extraction attenuated the inhibitory activity against trichothecene production. In addition, extraction of the non-autoclaved complex media with ethyl acetate, followed by removal of the solvent fraction, similarly resulted in increased accumulation of the mycotoxin. Although the increase in trichothecene production differed considerably among fungal strains and yeast extract products, F. graminearum species complex generally responded to the medium treatments in the same way. These results suggest that some hydrophobic substances that arise during the drying and heating of yeast extract negatively affected trichothecene production in liquid culture. Modes of actions of inhibitory substances were partially characterized using strain JCM 9873, with focus on the transcriptional and functional analyses of Tri6, a key regulator gene in trichothecene biosynthesis. The presence of the ethyl acetate-extractable substances in autoclaved YF_60 media decreased the relative transcription level of Tri6, as well as that of a trichodiene synthase gene Tri5. Thus, the substances exerted their inhibitory action through suppression of Tri6 expression. By using a yeast extract lot that completely prevented trichothecene production by the wild-type strain in autoclaved YS_60 medium, we prepared YF_60 media and cultured a constitutive Tri6 overexpressor strain described by Maeda et al. (2018). Despite the high transcription level of Tri6, the presence of the ethyl acetate extractable-substances suppressed 15-ADON production. These results suggested that both Tri6p-independent initial activation of Tri6 expression and subsequent Tri6p-dependent activation of Tri expression were affected by the hydrophobic substances in the yeast extract products.

Due to increasing anthropogenic impacts on deep-sea hydrothermal vent ecosystems, it is essential to understand population structure and maintenance through larval recruitment and recovery of vent faunas after disturbances. In this study, we quantified vent animal recruitment in the Okinawa Trough, in the western Pacific Ocean. This is the first study to investigate recruitment patterns at a man-made hydrothermal vent. Colonization plates were deployed at three sites. Site 1 manifested new hydrothermal shimmering with small chimneys, white bacterial mats, and some alvinocaridid shrimp that arrived after drilling. Site 2 showed no evidence of newly arrived foundation species after drilling, and Site 3 had pre-existing animal communities in the vicinity of the new vent. Twenty-two months after deployment, colonization plates were retrieved and recruited animals were inventoried. Species composition and abundance differed among sites, but relatively high similarity in species composition was observed at Sites 1 and 3, though not at Site 2. Newly established communities on the plates at Sites 1 and 2 (no pre-existing fauna) showed lower species richness and abundance than at Site 3. Differences in abundance and size-frequency distributions of major recruits on the plates (i.e. Lepetodrilus nux , Bathymodiolus spp.) suggest the importance of reproductive and early life-history characteristics in spatial variability of recruitment. Lepetodrilus nux populations established on the plates at Site 1 showed high genetic connectivity. These results illustrate the importance of localized recruitment, which may have a significant impact on sustainability of vent faunal populations, despite the existence of regional metapopulations.

Anthropogenic sources of arsenic poses and creates unintentional toxico-pathological concerns to humans in many parts of the world. The understanding of toxicity of this metalloid, which shares properties of both metal and non-metal is principally structured on speciation types and holy grail of toxicity prevention. Visible symptoms of arsenic toxicity include nausea, vomiting, diarrhea and abdominal pain. In this review, we focused on the dermal cell stress caused by trivalent arsenic trioxide and pentavalent arsanilic acid. Deciphering the molecular events involved during arsenic toxicity and signaling cascade interaction is key in arsenicosis prevention. FoxO1 and FoxO2 transcription factors, members of the Forkhead/Fox family, play important roles in this aspect. Like Foxo family proteins, ATM/CHK signaling junction also plays important role in DNA nuclear factor guided cellular development. This review will summarize and discuss current knowledge about the interplay of these pathways in arsenic induced dermal pathogenesis.

Population genetics of the coral genus Pocillopora have been more intensively studied than those of any other reef-building taxon. However, recent investigations have revealed that the current morphological classification is inadequate to represent genetic lineages. In this study, we isolated and characterized novel microsatellite loci from morphological Pocillopora meandrina (Type 1) and Pocillopora acuta (Type 5). Furthermore, we characterized previously reported microsatellite loci. A total of 27 loci (13 novel loci) proved useful for population genetic analyses at two sites in the Ryukyu Archipelago, in the northwestern Pacific. Clonal diversity differed in each genetic lineage. Genetic structure suggested by microsatellites corresponded to clusters in a phylogenetic tree constructed from a mitochondrial open reading frame (mtORF). In addition, we found an unknown mitochondrial haplotype of this mtORF. These microsatellite loci will be useful for studies of connectivity and genetic diversity of Pocillopora populations, and will also support coral reef conservation.

Trichothecene mycotoxins often accumulate in apparently normal grains of cereal crops. In an effort to develop an agricultural chemical to reduce trichothecene contamination, we screened trichothecene production inhibitors from the compounds on the chemical arrays. By using the trichodiene (TDN) synthase tagged with hexahistidine (rTRI5) as a target protein, 32 hit compounds were obtained from chemical library of the RIKEN Natural Product Depository (NPDepo) by chemical array screening. At 10 μg mL^− 1, none of the 32 chemicals inhibited trichothecene production by Fusarium graminearum in liquid culture. Against the purified rTRI5 enzyme, however, NPD10133 [progesterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine] showed weak inhibitory activity at 10 μg mL^− 1 (18.7 μM). For the screening of chemicals inhibiting trichothecene accumulation in liquid culture, 20 analogs of NPD10133 selected from the NPDepo chemical library were assayed. At 10 μM, only NPD352 [testosterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine methyl ester] inhibited rTRI5 activity and trichothecene production. Kinetic analysis suggested that the enzyme inhibition was of a mixed-type. The identification of NPD352 as a TDN synthase inhibitor lays the foundation for the development of a more potent inhibitor via systematic introduction of wide structural diversity on the gonane skeleton and amino acid residues.

Recent genetic research has begun to reveal population structures of deep-sea, hydrothermal vent species, but detailed assessments of genetic diversity and connectivity in hydrothermal vent populations, based on multiple genetic loci, are still scarce, especially in the Northwest Pacific. Accordingly, we isolated 38 novel polymorphic microsatellite loci from the limpet, Lepetodrilus nux, and 14 from the squat lobster, Shinkaia crosnieri, two dominant hydrothermal vent species, using next-generation sequencing. These loci revealed polymorphism levels of 5–20 alleles per locus in L. nux and 5–25 in S. crosnieri. Observed and expected heterozygosities ranged from 0.240 to 0.960 and 0.283 to 0.938 in L. nux and from 0.450 to 0.950 and 0.620 to 0.941 in S. crosnieri, respectively. Twelve loci in L. nux and four loci in S. crosnieri showed significant deviation from Hardy–Weinberg equilibrium (p

The elucidation of species diversity and connectivity is essential for conserving coral reef communities and for understanding the characteristics of coral populations. To assess the species diversity, intraspecific genetic diversity, and genetic differentiation among populations of the brooding coral Seriatopora spp., we conducted phylogenetic and population genetic analyses using a mitochondrial DNA control region and microsatellites at ten sites in the Ryukyu Archipelago, Japan. At least three genetic lineages of Seriatopora (Seriatopora-A, -B, and -C) were detected in our specimens. We collected colonies morphologically similar to Seriatopora hystrix, but these may have included multiple, genetically distinct species. Although sexual reproduction maintains the populations of all the genetic lineages, Seriatopora-A and Seriatopora-C had lower genetic diversity than Seriatopora-B. We detected significant genetic differentiation in Seriatopora-B among the three populations as follows: pairwise F ST = 0.064–0.116 (all P = 0.001), pairwise G′′ST = 0.107–0.209 (all P = 0.001). Additionally, only one migrant from an unsampled population was genetically identified within Seriatopora-B. Because the peak of the settlement of Seriatopora larvae is within 1 d and almost all larvae are settled within 5 d of spawning, our observations may be related to low dispersal ability. Populations of Seriatopora in the Ryukyu Archipelago will probably not recover unless there is substantial new recruitment from distant populations.

Fusarium species are traditionally grouped into type A and type B trichothecene producers based on structural differences in the mycotoxin they synthesize. The type B trichothecene-producing Fusarium graminearum strains are further divided into 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV) chemotypes. The former two chemotypes, collectively termed a deoxynivalenol (DON) chemotype, evolved from a NIV chemotype by inactivation of FgTri13, which encodes trichothecene C-4 hydroxylase, during the evolutionary process. Despite stable overexpression of FgTri13, however, both 3-acetylnivalenol (3-ANIV) and 3-ADON accumulate equally in shake flask culture of a transgenic 3-ADON chemotype. In this study, we investigated why the "3-ANIV chemotype" could not be obtained using this strategy. When analysis was extended to the transgenic NIV chemotype, in which FgTri7 C-4 acetylase gene was disrupted and FgTri8 deacetylase gene was replaced with the 3-ADON chemotype's orthologue, C-4 unoxygenated 3-ADON, as well as C-4 oxygenated 3-ANIV, accumulated as the end product. A feeding experiment with an ΔFgtri5ΔFgtri3 double gene disruptant, a trichothecene non-producing mutant unable to acetylate C-15 of the trichothecene ring, revealed the importance of the 15-O-acetyl group for efficient C-4 hydroxylation of DON-type trichothecenes. This implies that traditional DON and NIV chemotype diversification is not solely explained by FgTri13, but is also explained by the function of the FgTri8 trichothecene deacetylase gene. None of the crude cell extracts from existing chemotypes showed highly specific C-15 deacetylation activity against 3,15-diacetylnivalenol (3,15-diANIV) without deacetylating C-15 of the C-4 unoxygenated earlier intermediate, 3,15-diacetyldeoxynivalenol. Thus, an unnatural Fusarium trichothecene, 3-ANIV, could only be synthesized as part of a mixture with 3-ADON, unless the esterase encoded by FgTri8 evolves to act on the 15-O-acetyl of 3,15-diANIV with high specificity. We also explain why the transgenic "15-ANIV chemotype", which can be generated through functional inactivation of FgTri7, uses an engineered pathway via 3,15-diANIV, but not 15-ADON, to generate 15-ANIV. Tri genes appear to evolve continuously, and altered functions of trichothecene pathway enzymes result in the generation of new trichothecenes, such as NX-2 and NX-3, which have been recently discovered in field isolates of F. graminearum. As recombination of FgTri8 between existing F. graminearum isolates could give rise to a strain that produces mixtures of DON and NIV-type trichothecenes, it may also be noteworthy to monitor the emergence of a field isolate that invalidates traditional chemotype classification.

In this era of global climate change, understanding fundamental mechanisms of coral community maintenance and persistence in temperate non-reefal areas is a high marine conservation priority. To identify mechanisms of community maintenance and persistence via larval supply, we monitored coral settlement over 12 years and investigated the genetic population structure of two major acroporid species at Kushimoto, Wakayama Prefecture, Japan (33°N). Between 8 and 30 artificial settlement panel pairs were deployed from May or June to September, October, or November of each year. Recruits on settlement panel pairs were scarce, especially those of acroporids (0 or

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), induces disease resistance to the Fusarium head blight fungus Fusarium graminearum in Arabidopsis and barley, but it is unknown at which stage of the infection it acts. Since the rate of haustorial formation of an obligate biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was significantly reduced in NMN-treated coleoptile epidermal cells, the possibility that NMN induces resistance to the biotrophic stage of F. graminearum was investigated. The results show that NMN treatment caused the wandering of hyphal growth and suppressed the formation of appressoria-like structures. Furthermore, we developed an experimental system to monitor the early stage of infection in real-time and analyzed the infection behavior. We observed that the hyphae elongated windingly by NMN treatment. These results suggest that NMN potentiates resistance to the biotrophic invasion of F. graminearum as well as Bgh.

Seagrasses are marine angiosperms and play an essential ecological role in coastal ecosystems; however, seagrass meadows are threatened locally by anthropogenic disturbances. Understanding the dispersal patterns of seagrasses is essential for appropriate ecosystem management and establishment of marine protected areas (MPAs) in coastal ecosystems. In the Guimaras Strait in the Philippines, Banate (BAN) has been established as an MPA. However, there is a lack of information on the genetic diversity of seagrasses in BAN and the surrounding areas. In the present study, population genetics analysis of Enhalus acoroides was performed by using polymorphic microsatellite markers, for the estimation of genetic diversity, differentiation, and migration patterns of seagrasses within the regional geographical scale (~200 km) around the Guimaras Strait. The results showed that the genetic diversity of BAN is extremely low, although the Guimaras Strait is located in the tropical central habitat. Guimaras Island geographically divides the populations of E. acoroides into south and north. However, the genetic structure did not show any relationship between the geographical location and distance. The floating, buoyant fruits of E. acoroides may play a role in their long-distance dispersal; however, such dispersal is not frequent. Almost all of the seeds and fruits are derived from self-recruitment in the natal meadow. This study suggests that E. acoroides populations possess a weak genetic connectivity, and that the persistence of the meadow is threatened due to the low genetic diversity and high degree of population isolation in BAN. To maintain and enhance the genetic diversity of seagrasses within the MPA, the seagrass meadows in the surrounding areas should also be conserved.

Fructo-oligosaccharides containing a sucrose unit are reported as carbon sources necessary for trichothecene production by Fusarium graminearum. Here we demonstrate that trichothecene production is induced when at least 100μM sucrose is added to a culture medium containing 333mM glucose in a 24-well plate. When glucose, the main carbon source of the medium, was replaced with galactose, maltose, or sorbitol, the addition of 100μM sucrose could no longer induce trichothecene production. However, replacing half the amount of each carbon source with glucose restored the trichothecene production-inducing activity of sucrose. Detailed investigations with media containing various concentrations of galactose and glucose as carbon sources suggested that operation of the galactose catabolic pathway for energy conservation affected trichothecene biosynthesis induction by sucrose. Trichothecene production was also induced by 100μM of either raffinose or xylosucrose in axenic liquid culture medium containing glucose as the major carbon source. These results demonstrate that sucrose derivatives are not necessary as a carbon source for inducing trichothecene biosynthesis, and that the minimum structural requirement for sugars to function as trichothecene production-inducer molecules is to contain an α-(1→2) (glucosyl/xylosyl)-fructosyl linkage.

Tricholoma matsutake is an economically valuable fungus in Japan. Its fruit bodies occur in colonies established in natural pine forests. We traced colony establishment processes of T. matsutake in 2001 and 2010 by using microsatellite markers. T. matsutake genets persisted at least for a decade. Comparing colonies found in 2001 and 2010, five novel colonies were observed in 2010, where the organic layer was removed or pine trees were regenerated less than 50 y ago. The novel colonies were genotyped, and all of them were different from colonies found in 2001, indicating that these colonies were established by dispersed basidiospores. Novel genet and candidate parent genets were located in the same ridge, and the mean geographical distance between a novel genet and a parent genet was 19.59 ± 14.73 m, indicating that basidiospores may colonize more easily on roots of hosts near parental basidiocarps. In conclusion, sufficient basidiospore supply and organic layer removal or pine tree regeneration were essential factors for colony establishment of T. matsutake.

To establish effective locations and sizes of potential protected areas for reef ecosystems, detailed information about source and sink relationships between populations is critical, especially in archipelagic regions. Therefore, we assessed population structure and genetic diversity of Acropora tenuis, one of the dominant stony coral species in the Pacific, using 13 microsatellite markers to investigate 298 colonies from 15 locations across the Nansei Islands in southwestern Japan. Genetic diversity was not significant among sampling locations, even in possibly peripheral locations. In addition, our results showed that there are at least two populations of A. tenuis in the study area. The level of genetic differentiation between these populations was relatively low, but significant between many pairs of sampling locations. Directions of gene flow, which were estimated using a coalescence‐based approach, suggest that gene flow not only occurs from south to north, but also from north to south in various locations. Consequently, the Yaeyama Islands and the Amami Islands are potential northern and southern sources of corals. On the other hand, the Miyako Islands and west central Okinawa Island are potential sink populations. The Kerama Islands and the vicinity of Taketomi Island are potential contact points of genetic subdivision of coral populations in the Nansei Islands. We found that genetic population structure of A. tenuis in the Nansei Islands is more complex than previously thought. These cryptic populations are very important for preserving genetic diversity and should be maintained.

Fusarium sporotrichioides genes FsTri11, FsTri13, and FsTri1 encode cytochrome P450 monooxygenases (CYPs) responsible for hydroxylations at C-15, C-4, and C-8 of the trichothecene skeleton, respectively. However, the corresponding genes of nivalenol (NIV)-chemotype Fusarium graminearum remain to be functionally elucidated. In this study, we characterized the roles of these CYPs in NIV biosynthesis. Analyses of the metabolites of the F. graminearum Fgtri11- mutant, a disruptant of FgTri11 encoding isotrichodermin (ITD) C-15 hydroxylase, revealed a small amount of NIV-type trichothecenes suggesting that an alternative C-15 hydroxylase partially complemented FgTRI11p. In contrast, the C-7/C-8 hydroxylations depended solely on FgTRI1p, as suggested by the metabolite profiles of the Fgtri11- Fgtri1- double gene disruptant. Disruption of FgTri1 in both the wild-type and Fgtri13- mutant backgrounds revealed that FgTRI13p exhibits marginal activity toward calonectrin (CAL) and that it was the only C-4 hydroxylase. In addition, feeding experiments demonstrated that the C-4 hydroxylation of a 7-hydroxytrichothecene lacking C-8 ketone was extremely limited. The marginal activity of FgTRI13p toward CAL was advantageous for the C-7/C-8 hydroxylation steps in NIV biosynthesis, as transformation of a C-4 oxygenated trichothecene lacking C-7/C-8 modifications into NIV-type trichothecenes was quite inefficient. The significance of hydroxylation steps in the evolution of Fusarium trichothecenes is discussed.

Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.

Stony corals (Scleractinia) form the basis for some of the most diverse ecosytems on Earth, but we have much to learn about their evolutionary history and systematic relationships. In order to improve our understanding of species in corals we here investigated phylogenetic relationships between morphologically defined species and genetic lineages in the genus Galaxea (Euphyllidae) using a combined phylogenomic and phylogeographic approach. Previous studies revealed the nominal species G. fascicularis included three genetically well-differentiated lineages (L, S & L+) in the western Pacific, but their distribution and relationship to other species in the genus was unknown. Based on genomic (RAD-seq) and mitochondrial sequence data (non-coding region between cytb and ND2) we investigated whether the morphological taxa represent genetically coherent entities and what is the phylogenetic relationship and spatial distribution of the three lineages of G. fascicularis throughout the observed species range. Using the RAD-seq data, we find that the genus Galaxea is monophyletic and contains three distinct clades: an Indo-Pacific, a Pacific, and a small clade restricted to the Chagos Archipelago. The three lineages of G. fascicularis were associated with different RAD-seq clades, with the ‘L’ lineage showing some morphological distinction from the other two lineages (larger more asymmetrical polyps). In addition to these, three more genetic lineages in G. fascicularis may be distinguished – a Chagossian, an Ogasawaran, and one from the Indian-Red Sea. Among nominal taxa for which we have multiple samples, G. horrescens was the only monophyletic species. The mitochondrial non-coding region is highly conserved apart of the length polymorphism used to define L, S & L+ lineages and lacks the power to distinguish morphological and genetic groups resolved with genomic RAD-sequencing. The polyphyletic nature of most species warrants a careful examination of the accepted taxonomy of this group with voucher collections and their comparison to type specimens to resolve species boundaries. Further insight to the speciation process in corals will require international cooperation for the sharing of specimens to facilitate scientific discovery.

Populations of marine organisms on coral reef islands (CRI) are connected in space and time by seawater that transports propagules of plants, animals, and algae. Yet, despite this reality, it is often assumed that routine replenishment of populations of marine organisms on CRI is supported by locally-sourced propagules (hereafter, larvae). Following large disturbances, however, distantly-sourced larvae from less disturbed CRI within a regional meta-population are likely to be important for local population recovery, but evaluating the roles of locally- vs. distantly-sourced larvae remains difficult. While larval sources are relatively well-known for some fishes, they remain virtually unknown for most taxa, particularly those associated with the benthos, as exemplified by scleractinian corals. This review focuses on reef recovery and larval connectivity. Using corals as examples, we argue that CRI can serve as natural laboratories in which studies of these issues can enhance understanding of coral reef community dynamics under future disturbance regimes. Rather than focusing on synthesizing empirical data, we focus on the capacity for CRI to realize their potential in this research area, concluding that progress is impeded by the limited breadth, detail, and spatio-temporal concordance of existing research. Using long-term observational programs of coral reefs in Mo'orea (French Polynesia), Okinawa (Japan), and St. John (US Virgin Islands) as examples of the data currently available, we make the case that new modes of multidisciplinary and collaborative research will be required to exploit the value of CRI in understanding the role of connectivity in mediating ecosystem resilience in a future affected by anthropogenic disturbances.

Nitrogen sources in media have a significant impact on the onset of secondary metabolism in fungi. For transcriptional activation of many nitrogen catabolic genes, an AreA transcription factor is indispensable. This also holds true for Fusarium graminearum that produces trichothecenes, an important group of mycotoxin, in axenic culture. Despite the presence of numerous consensus AreA-binding sites in the promoters of Tri genes in the trichothecene cluster core region, the effect of medium amino acids on trichothecene biosynthesis is poorly understood. In this study, we examined the effect of certain amino acids, which were predicted to activate AreA function and increase Tri gene transcription, on trichothecene production in liquid culture. By frequent monitoring and adjustments in the pH of the culture medium, including replacement of the spent medium with fresh medium, we demonstrate the suppressive effects of the amino acids, used as the sole nitrogen source, on trichothecene biosynthesis. When the medium pH was maintained at 4.0, Gly, L-Ser, and L-Thr suppressed trichothecene production by F. graminearum. Enhanced trichothecene-inducing effects were observed when the medium pH was 3.5, with only L-Thr suppressing trichothecene synthesis.

Osteopenic disorders such as osteoporosis and rheumatoid arthritis are characterized by excessive bone resorption by osteoclasts relative to bone formation by osteoblasts. MicroRNAs are emerging as key players in bone remodeling, modulating the functions of both osteoblasts and osteoclasts. Among them, miR-21 is highly expressed in osteoclast precursors and is known to regulate genesis, differentiation, and apoptosis of osteoclasts. The pro-osteoclastogenic nature of miR-21 makes it a potential candidate as a therapeutic target to treat bone disorders. We had previously demonstrated that anthroglycoside aloin derived from Aloe vera was effective in promoting osteoblastogenesis and inhibiting osteoclastogenesis. The present study investigated the role of miR-21 in aloin’s inhibitory effect on osteoclast differentiation. Aloin effectively suppressed receptor activator of nuclear factor kappa-B (NFĸB) ligand (RankL)-induced miR-21 expression via repression of NFĸB activation. MiR-21 suppression resulted in upregulation of osteoclast suppressor programmed cell death protein 4 (PDCD4), and downregulation of osteoclast marker cathepsin K. Knockdown or gain-of-function studies revealed that miR-21 was pivotal to aloin’s inhibitory effect on osteoclastogenesis. This study also highlights the dynamic potential of aloin as a therapeutic agent to treat osteopenic disorders.

An artificial metabolic route to an unnatural trichothecene was designed by taking advantage of the broad substrate specificities of the T-2 toxin biosynthetic enzymes of Fusarium sporotrichioides. By feeding 7-hydroxyisotrichodermin, a shunt pathway metabolite of F. graminearum, to a trichodiene synthase-deficient mutant of F. sporotrichioides, 7-hydroxy T-2 toxin (1) was obtained as the final metabolite. Such an approach may have future applications in the metabolic engineering of a variety of fungal secondary metabolites. The toxicity of 7-hydroxy T-2 toxin was 10 times lower than that of T-2 toxin in HL-60 cells.

From the RIKEN Natural Products Depository (NPDepo) chemical library, we identified small molecules that alter trichothecene 15-acetyldeoxynivalenol (15-ADON) production by Fusarium graminearum. Among trichothecene production activators, a furanocoumarin NPD12671 showed the strongest stimulatory activity on 15-ADON production by the fungus cultured in a 24-well plate. NPD12671 significantly increased the transcription of Tri6, a transcription factor gene necessary for trichothecene biosynthesis, in both trichothecene-inducing and noninducing culture conditions. Dihydroartemisinin (DHA) was identified as the most effective inhibitor of trichothecene production in 24-well plate culture; DHA inhibited trichothecene production (50% inhibition at 1 μM) without affecting fungal mass by suppressing Tri6 expression. To determine the effect of DHA on trichothecene pathway Tri gene expression, we generated a constitutively Tri6-overexpressing strain that produced 15-ADON in YG_60 medium in Erlenmeyer flasks, conditions under which no trichothecenes are produced by the wild-type. While 5 μM DHA failed to inhibit trichothecene biosynthesis by the overexpressor in trichothecene-inducing YS_60 culture, trichothecene production was suppressed in the YG_60 culture. Regardless of a high Tri6 transcript level in the constitutive overexpressor, the YG_60 culture showed reduced accumulation of Tri5 and Tri4 mRNA upon treatment with 5 μM DHA. Deletion mutants of FgOs2 were also generated and examined; both NPD12671 and DHA modulated trichothecene production as they did in the wild-type strain. These results are discussed in light of the mode of actions of these chemicals on trichothecene biosynthesis.

Disruption of two Fusarium genes that negatively regulate trichothecene biosynthesis was reported to cause a drastic increase in trichothecene production. However, careful inspection of these genes revealed that neither was significantly related to trichothecene production. Agmatine medium maintained the expression of trichothecene genes at significant levels, resulting in a 2–3-fold increase in the final yield, as compared to glutamine medium.

Population connectivity resulting from larval dispersal is essential for the maintenance or recovery of populations in marine ecosystems, including coral reefs. Studies of species diversity and genetic connectivity within species are essential for the conservation of corals and coral reef ecosystems. We analyzed mitochondrial DNA sequence types and microsatellite genotypes of the broadcast‐spawning coral, Galaxea fascicularis, from four regions in the subtropical Nansei Islands in the northwestern Pacific Ocean. Two types (soft and hard types) of nematocyst morphology are known in G. fascicularis and are significantly correlated with the length of a mitochondrial DNA noncoding sequence (soft type: mt‐L; hard type: mt‐S type). Using microsatellites, significant genetic differentiation was detected between the mitochondrial DNA sequence types in all regions. We also found a third genetic cluster (mt‐L+), and this unexpected type may be a cryptic species of Galaxea. High clonal diversity was detected in both mt‐L and mt‐S types. Significant genetic differentiation, which was found among regions within a given type (F ST = 0.009–0.024, all Ps ≤ 0.005 in mt‐L; 0.009–0.032, all Ps ≤ 0.01 in mt‐S), may result from the shorter larval development than in other broadcast‐spawning corals, such as the genus Acropora. Nevertheless, intraspecific genetic diversity and connectivity have been maintained, and with both sexual and asexual reproduction, this species appears to have a potential for the recovery of populations after disturbance.

Arsenic exposure through drinking water is a major public health problem. It causes a number of toxic effects on skin. Arsenic has been reported to inhibit cell proliferation in in vitro conditions. However, reports about the molecular mechanisms are limited. Here, we investigated the mechanism involved in arsenic acid-mediated inhibition of cell proliferation using mouse skin fibroblast cell line. The present study found that 10 ppm arsenic acid inhibited cell proliferation, without any effect on cell death. Arsenic acid induced the generation of reactive oxygen species (ROS), resulting in oxidative stress to DNA. It also activated the mammalian Ste20-like protein kinase 1 (MST1); however the serine/threonine kinase Akt was downregulated. Forkhead box O (FOXO) transcription factors are activated through phosphorylation by MST1 under stress conditions. They are inhibited by phosphorylation by Akt through external and internal stimuli. Activation of FOXOs results in their nuclear localization, followed by an increase in transcriptional activity. Our results showed that arsenic induced the nuclear translocation of FOXO1 and FOXO3a, and altered the cell cycle, with cells accumulating at the G2/M phase. These effects caused cellular senescence. Taken together, our results indicate that arsenic acid inhibited cell proliferation through cellular senescence process regulated by MST1-FOXO signaling pathway.

Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) caused by a mycotoxigenic fungus Fusarium graminearum resulting in significantly decreased yields and accumulation of toxic trichothecenes in grains. We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.

Recently, seagrasses have been declining along coastlines worldwide. We genetically analyzed 1058 ramets of Syringodium isoetifolium collected from 21 populations in the Philippines and 9 in the Ryukyu Islands using nine microsatellite markers, to provide a baseline of genetic information on seagrasses in the western North Pacific region under the influence of the Kuroshio Current, and aid in their genetic conservation. We detected 421 genotypes and the average clonal richness (R) was relatively low (R = 0.38), suggesting that S. isoetifolium populations were formed mainly through asexual reproduction. The index of genetic differentiation among populations for S. isoetifolium was high ( $$D_{ST}^{\prime }$$ = 0.238 and $$G_{ST}^{\prime }$$ = 0.337), and most pairwise F ST values were significantly greater than zero, suggesting that gene flow among populations is rare. Clear genetic structure in S. isoetifolium populations were detected, indicating four genetically-similar bioregions, that is, the Ryukyu, northwest Philippines, northeast Philippines, and central Philippines populations. This genetic structure indicated that the Kuroshio strongly affects the formation of S. isoetifolium populations and should be factored into conservation efforts for S. isoetifolium.

Persistence of populations at their distributional ranges relies on local population dynamics and the fitness of species with low dispersal potential. We analyzed the population genetic diversity and structure of a tropical seagrass species, Cymodocea serrulata, at 34 sites spanning Philippine (diversity hotspot) and Ryukyu Islands (northern limit of distribution) populations using microsatellite (SSR) markers. Seagrass populations in the diversity hotspot are hypothesized to contain higher genetic diversity and clonal richness than those resulting from expansion or geographic range limits. We tested this hypothesis by comparing the genetic diversity, genetic structure and clonal richness of C. serrulata populations in the Philippines and Ryukyu Islands. C. serrulata populations showed decreased genetic diversity and clonal richness at their northern limit. Clonal reproduction predominated at the northern limit, while sexual reproduction prevailed in the diversity hotspot. Decreased genetic diversity and clonal richness at the northern limit may be the consequence of drift resulting from founder effect, reduced habitat, sea surface temperature and low gene flow and/or natural selection across life stages, wherein clonal reproduction confers greater environmental fitness. Analysis of molecular variance (AMOVA) and the fixation index, F ST, showed significant genetic differentiation within and among geographic populations. STRUCTURE analysis revealed that the Ryukyu Islands populations were mosaics of genets from the eastern Philippines, likely carried by the Kuroshio Current.

Understanding how coral populations are established and maintained is important to predict how coral reef ecosystems will respond to future conditions. Population genetic analyses using DNA markers have provided useful information on how coral populations are maintained. In this chapter, I briefly introduce the history of using DNA markers in the population genetic analyses of corals. I also explain the merit of population genetic analyses to delineate the species boundaries of corals and infer how reproductive characteristics contribute to connectivity among populations. Based on previous studies on population genetic analyses of corals, I also discuss how population genetic analyses have contributed toward understanding the patterns of connectivity among coral populations and geographic variations in genetic diversity, primarily focusing on examples along the Ryukyu Archipelago. Finally, I propose future directions for the population genetics of corals in Japan, taking several aspects into consideration, including methodological information, such as seascape genetics and the development of novel molecular markers for utilizing next-generation sequencing technologies.

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium to invade rice cells. In this report, we show that CBP1, which encodes a chitin-deacetylase, is involved in the induction phase of appressorium differentiation. We demonstrate that the enzymatic activity of Cbp1 is critical for appressorium formation. M. oryzae has six CDA homologues in addition to Cbp1, but none of these are indispensable for appressorium formation. We observed chitosan localization at the fungal cell wall using OGA488. This observation suggests that Cbp1-catalysed conversion of chitin into chitosan occurs at the cell wall of germ tubes during appressorium differentiation by M. oryzae. Taken together, our results provide evidence that the chitin deacetylase activity of Cbp1 is necessary for appressorium formation.

Information on genetic diversity and differentiation of seagrass populations is essential for the conservation of coastal ecosystems. However, little is known about the seagrasses in the Indo-West Pacific Ocean, where the world's highest diversity of seagrasses occurs. The influence of sea currents on these populations is also unknown. We estimated the genetic diversity and population genetic structure and identified reproductive features in Enhalus acoroides populations from the Yaeyama Islands, Hainan Island and the Philippines. The Philippines are situated at the centre of the E. acoroides range, Yaeyama and Hainan are peripheral populations, and the Yaeyama population is at the northern limit of the species range. The powerful Kuroshio Current flows from the Philippines to Yaeyama. Genetic analyses using nine microsatellite markers indicated that reproduction of E. acoroides is mostly sexual. Clonal diversity does not decrease in northern populations, although genetic diversity does. However, the genetic diversity of the Yaeyama populations is greater than that of the Hainan populations. Significant genetic differentiation among most populations was evident; however, the Yaeyama and north-east Philippines populations were genetically similar, despite being separated by ~1100 km. An assignment test suggested that recruitment occurs from the north-east Philippines to Yaeyama. The strong current in this region is probably responsible for the extant genetic diversity and recruitment patterns.

Transforming growth factor beta1 (TGFβ1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFβ1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGFβ1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFβ1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGFβ1 in high glucose conditions. The induction by TGFβ1 was dependent on NFκB activation and subsequent ROS generation. TGFβ1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFβ1. ChIP assay revealed binding of p65 to miR-21 promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGFβ1-NFκB-miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.

Barnacles of the genus Neoverruca are abundant near deep-sea hydrothermal vents of the northwestern Pacific Ocean, and are useful for understanding processes of population formation and maintenance of deep-sea vent faunas. Using next-generation sequencing, we isolated 12 polymorphic microsatellite loci from Neoverruca sp., collected in the Okinawa Trough. These microsatellite loci revealed 2–19 alleles per locus. The expected and observed heterozygosities ranged from 0.286 to 1.000 and 0.349 to 0.935, respectively. Cross-species amplification showed that 9 of the 12 loci were successfully amplified for Neoverruca brachylepadoformis in the Mariana Trough. A pairwise FST value calculated using nine loci showed significant genetic differentiation between the two species. Consequently, the microsatellite markers we developed will be useful for further population genetic studies to elucidate genetic diversity, differentiation, classification, and evolutionary processes in the genus Neoverruca.

Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 μmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.