A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Regulatory mechanism of trichothecene biosynthesis in Fusarium graminearum.

Among the genes involved in the biosynthesis of trichothecene (Tri genes), Tri6 and Tri10 encode a transcription factor with unique Cys2His2 zinc finger domains and a regulatory protein with no consensus DNA-binding sequences, respectively. Although various chemical factors, such as nitrogen nutrients, medium pH, and certain oligosaccharides, are known to influence trichothecene biosynthesis in Fusarium graminearum, the transcriptional regulatory mechanism of Tri6 and Tri10 genes is poorly understood. Particularly, culture medium pH is a major regulator in trichothecene biosynthesis in F. graminearum, but it is susceptible to metabolic changes posed by nutritional and genetic factors. Hence, appropriate precautions should be considered to minimize the indirect influence of pH on the secondary metabolism while studying the roles of nutritional and genetic factors on trichothecene biosynthesis regulation. Additionally, it is noteworthy that the structural changes of the trichothecene gene cluster core region exert considerable influence over the normal regulation of Tri gene expression. In this perspective paper, we consider a revision of our current understanding of the regulatory mechanism of trichothecene biosynthesis in F. graminearum and share our idea toward establishing a regulatory model of Tri6 and Tri10 transcription.

Fluro-Protein C-Phycocyanin Docked Silver Nanocomposite Accelerates Cell Migration through NFĸB Signaling Pathway.

Currently, there is a great demand for the development of nanomedicine aided wound tissue regeneration via silver doped nanoceuticals. Unfortunately, very little research is being carried out on antioxidants-doped silver nanometals and their interaction on the signaling axis during the bio-interface mechanism. In this study, c-phycocyanin primed silver nano hybrids (AgcPCNP) were prepared and analyzed for properties such as cytotoxicity, metal decay, nanoconjugate stability, size expansion, and antioxidant features. Fluctuations in the expression of marker genes during cell migration phenomena in in vitro wound healing scenarios were also validated. Studies revealed that physiologically relevant ionic solutions did not exhibit any adverse effects on the nanoconjugate stability. However, acidic, alkali, and ethanol solutions completely denatured the AgcPCNP conjugates. Signal transduction RT2PCR array demonstrated that genes associated with NFĸB- and PI3K-pathways were significantly (p < 0.5%) altered between AgcPCNP and AgNP groups. Specific inhibitors of NFĸB (Nfi) and PI3K (LY294002) pathways confirmed the involvement of NFĸB signaling axes. In vitro wound healing assay demonstrated that NFĸB pathway plays a prime role in the fibroblast cell migration. In conclusion, the present investigation revealed that surface functionalized AgcPCNP accelerated the fibroblast cell migration and can be further explored for wound healing biomedical applications.

Genomic analysis of a reef-building coral, Acropora digitifera, reveals complex population structure and a migration network in the Nansei Islands, Japan.

Understanding the structure and connectivity of coral populations is fundamental for developing marine conservation policies, especially in patchy environments such as archipelagos. The Nansei Islands, extending more than 1000 km in southwestern Japan, are characterized by high levels of biodiversity and endemism, supported by coral reefs, which make this region ideal for assessing genetic attributes of coral populations. In this study, we conducted population genomic analyses based on genome-wide, single-nucleotide polymorphisms (SNPs) of Acropora digitifera, a common species in the Nansei Islands. By merging newly obtained genome resequencing data with previously published data, we identified more than 4 million genome-wide SNPs in 303 colonies collected at 22 locations, with sequencing coverage ranging from 3.91× to 27.41×. While population structure analyses revealed genetic similarities between the southernmost and northernmost locations, separated by >1000 km, several subpopulations in intermediate locations suggested limited genetic admixture, indicating conflicting migration tendencies in the Nansei Islands. Although migration networks revealed a general tendency of northward migration along the Kuroshio Current, a substantial amount of southward migration was also detected, indicating important contributions of minor ocean currents to coral larval dispersal. Moreover, heterogeneity in the transition of effective population sizes among locations suggests different histories for individual subpopulations. The unexpected complexity of both past and present population dynamics in the Nansei Islands implies that heterogeneity of ocean currents and local environments, past and present, have influenced the population structure of this species, and similar unexpected population complexities may be expected for other marine species with similar reproductive modes.

Retinoic acid attenuates nuclear factor kappaB mediated induction of NLRP3 inflammasome.

BACKGROUND: Acetylcholine (ACh), a neurotransmitter and a part of the cholinergic system, can modify immune responses. Expression of acetylcholine receptors (AChR) in immune cells, including macrophages, leads to modulation of their function. Inflammasomes are part of the innate immune system and have been linked to a variety of inflammatory diseases. The NLRP3/ASC/caspase-1/IL-1 axis has emerged as a critical signaling pathway in inflammation process initiation. The role of ACh in modulating inflammasomes in macrophages remains relatively under-explored. METHODS: The effect of AChR agonist carbachol on inflammasome expression was investigated using murine and human macrophages. Cell lysates were assessed by western blot for protein analysis. Immunofluorescence studies were used to study the translocation of p65. The experiments were conducted in the presence of NF-ĸB inhibitor, AChR antagonists, and retinoic acid (RA) to study the role of NF-ĸB, ACh receptors, and RA, respectively. RESULTS: We found that carbachol increased the expression of NLRP3 inflammasome (NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18). The treated cells also showed an increase in NF-ĸB activation. The effect of carbachol was diminished by NF-ĸB inhibitor and atropine, a mAChR antagonist. The addition of RA also significantly reduced the effect of carbachol on NLRP3 inflammasomes. CONCLUSIONS: Our current study suggests that carbachol induces NLRP3 inflammasome activation through mAChR and NF-ĸB, and that RA abolishes the inflammatory response. It reveals the potentials of co-administration of RA with cholinergic drugs to prevent inflammatory responses during cholinergic medications.

The emerging role of exosomes in innate immunity, diagnosis and therapy.

Exosomes, which are nano-sized transport bio-vehicles, play a pivotal role in maintaining homeostasis by exchanging genetic or metabolic information between different cells. Exosomes can also play a vital role in transferring virulent factors between the host and parasite, thereby regulating host gene expression and the immune interphase. The association of inflammation with disease development and the potential of exosomes to enhance or mitigate inflammatory pathways support the notion that exosomes have the potential to alter the course of a disease. Clinical trials exploring the role of exosomes in cancer, osteoporosis, and renal, neurological, and pulmonary disorders are currently underway. Notably, the information available on the signatory efficacy of exosomes in immune-related disorders remains elusive and sporadic. In this review, we discuss immune cell-derived exosomes and their application in immunotherapy, including those against autoimmune connective tissue diseases. Further, we have elucidated our views on the major issues in immune-related pathophysiological processes. Therefore, the information presented in this review highlights the role of exosomes as promising strategies and clinical tools for immune regulation.

4-O-Glucosylation of Trichothecenes by Fusarium Species: A Phase II Xenobiotic Metabolism for t-Type Trichothecene Producers.

The t-type trichothecene producers Fusarium sporotrichioides and Fusarium graminearum protect themselves against their own mycotoxins by acetylating the C-3 hydroxy group with Tri101p acetylase. To understand the mechanism by which they deal with exogenously added d-type trichothecenes, the Δtri5 mutants expressing all but the first trichothecene pathway enzymes were fed with trichodermol (TDmol), trichothecolone (TCC), 8-deoxytrichothecin, and trichothecin. LC-MS/MS and NMR analyses showed that these C-3 unoxygenated trichothecenes were conjugated with glucose at C-4 by α-glucosidic linkage. As t-type trichothecenes are readily incorporated into the biosynthetic pathway following the C-3 acetylation, the mycotoxins were fed to the ΔFgtri5ΔFgtri101 mutant to examine their fate. LC-MS/MS and NMR analyses demonstrated that the mutant conjugated glucose at C-4 of HT-2 toxin (HT-2) by α-glucosidic linkage, while the ΔFgtri5 mutant metabolized HT-2 to 3-acetyl HT-2 toxin and T-2 toxin. The 4-O-glucosylation of exogenously added t-type trichothecenes appears to be a general response of the ΔFgtri5ΔFgtri101 mutant, as nivalenol and its acetylated derivatives appeared to be conjugated with hexose to some extent. The toxicities of 4-O-glucosides of TDmol, TCC, and HT-2 were much weaker than their corresponding aglycons, suggesting that 4-O-glucosylation serves as a phase II xenobiotic metabolism for t-type trichothecene producers.

Accumulation of 4-Deoxy-7-hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-Type FgTri1 Gene.

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.

Development of an in-situ forming, self-healing scaffold for dermal wound healing: in-vitro and in-vivo studies.

The importance of the extra-cellular matrix (ECM) for wound healing has been extensively researched. Understanding its importance, multiple ECM mimetic scaffolds have been developed. However, the majority of such scaffolds are prefabricated. Due to their stiffness, prefabricated scaffolds cannot come into direct contact with the basal skin cells at the wound bed, limiting their efficacy. We have developed a unique wound dressing, using chitosan (CH) and chondroitin sulfate (CS), that can form a porous scaffold (CH-CS PEC) in-situ, at the wound site, by simple mixing of the polymer solutions. As CH is positively and CS is negatively charged, mixing these two polymer solutions would lead to electrostatic cross-linking between the polymers, converting them to a porous, viscoelastic scaffold. Owing to the in-situ formation, the scaffold can come in direct contact with the cells at the wound bed, supporting their proliferation and biofunction. In the present study, we confirmed the cross-linked scaffold formation by solid-state NMR, XRD, and TGA analysis. We have demonstrated that the scaffold had a high viscoelastic property, with self-healing capability. Both keratinocyte and fibroblast cells exhibited significantly increased migration and functional markers expression when grown on this scaffold. In the rat skin-excisional wound model, treatment with the in-situ forming CH-CS PEC exhibited enhanced wound healing efficacy. Altogether, this study demonstrated that mixing CH and CS solutions lead to the spontaneous formation of a highly viscoelastic, porous scaffold, which can support epidermal and dermal cell proliferation and bio-function, with an enhanced in-vivo wound healing efficacy.

Nanoceutical Adjuvants as Wound Healing Material: Precepts and Prospects.

Dermal wound healing describes the progressive repair and recalcitrant mechanism of 12 damaged skin, and eventually, reformatting and reshaping the skin. Many probiotics, nutritional supplements, metal nanoparticles, composites, skin constructs, polymers, and so forth have been associated with the improved healing process of wounds. The exact mechanism of material-cellular interaction is a point of immense importance, particularly in pathological conditions such as diabetes. Bioengineered alternative agents will likely continue to dominate the outpatient and perioperative management of chronic, recalcitrant wounds as new products continue to cut costs and improve the wound healing process. This review article provides an update on the various remedies with confirmed wound healing activities of metal-based nanoceutical adjuvanted agents and also other nano-based counterparts from previous experiments conducted by various researchers.

Nicotinamide Mononucleotide Potentiates Resistance to Biotrophic Invasion of Fungal Pathogens in Barley.

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.

Retinoic acid abrogates LPS-induced inflammatory response via negative regulation of NF-kappa B/miR-21 signaling.

CONTEXT: Macrophages are essential components of the immune system, with significant roles in inflammation modulation. They can be activated into pro-inflammatory M1 or anti-inflammatory M2 phenotypes, depending on their micro-environment. Molecular factors that modulate macrophage polarization are hot targets for therapeutic strategies to counter chronic inflammatory pathological conditions. OBJECTIVE: The current study aimed to elucidate the molecular mechanisms by which Retinoic acid (RA), a potent immunomodulator, suppresses LPS-induced inflammatory response in macrophages. MATERIALS AND METHODS: RAW 264.7 macrophages were treated with RA and/or LPS, and analyzed for inflammatory genes and miR-21 by PCR. The roles of miR-21 and NF-ĸB signaling pathway were also assessed by knock-down experiments, immunofluorescence, and ChIP assays. RESULTS: Pretreatment with RA quenched the LPS-induced inflammatory responses, including phagocytosis, ROS generation, and NO production. RA shifted the polarization away from the M1 state by negative regulation of IKKα/ß, p65, and miR-21. RA hindered the phosphorylation of IKKα/ß, translocation of p65 into the nucleus, and the subsequent upregulation of miR-21. Knock-in and knock-down experiments showed that miR-21 is central for the polarization shift toward the pro-inflammatory M1 state. CONCLUSION: miR-21 is involved in the LPS-induced pro-inflammatory profile of macrophages and that RA negatively regulates the inflammatory response by targeting NF-ĸB/miR-21 signaling. Our data exposes RA's potential as a pharmacological agent to manipulate miR-21 and counteract hyper-inflammatory response.

Chromatic intervention and biocompatibility assay for biosurfactant derived from Balanites aegyptiaca (L.) Del.

Extraction of biosurfactants from plants is advantageous than from microbes. The properties and robustness of biosurfactant derived from the mesocarp of Balanites aegyptiaca have been reported. However, the dark brown property of biosurfactant and lack of knowledge of its biocompatibility limits its scope. In the present work, the decolorization protocol for this biosurfactant was optimized using hydrogen peroxide. The hemolytic potential and biocompatibility based on cell toxicity and proliferation were also investigated. This study is the first report on the decolorization and toxicity assay of this biosurfactant. For decolorization of biosurfactant, 34 full factorial design was used, and the data were subjected to ANOVA. Results indicate that 1.5% of hydrogen peroxide can decolorize the biosurfactant most efficiently at 40 °C in 70 min at pH 7. Mitochondrial reductase (MTT) and reactive oxygen species (ROS) assays on M5S mouse skin fibroblast cells revealed that decolorized biosurfactant up to 50 µg/mL for 6 h had no significant toxic effect. Hemolysis assay showed ~ 2.5% hemolysis of human RBCs, indicating the nontoxic effect of this biosurfactant. The present work established a decolorization protocol making the biosurfactant chromatically acceptable. Biocompatibility assays confirm its safer use as observed by experiments on M5S skin fibroblast cells under in vitro conditions.

MicroRNA 21 Elicits a Pro-inflammatory Response in Macrophages, with Exosomes Functioning as Delivery Vehicles.

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.

Impact of nitrogen metabolism-associated culture pH changes on regulation of Fusarium trichothecene biosynthesis: revision of roles of polyamine agmatine and transcription factor AreA.

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.

Evolutionary biogeography of the reef-building coral genus Galaxea across the Indo-Pacific ocean.

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.

c-Phycocyanin primed silver nano conjugates: Studies on red blood cell stress resilience mechanism.

Green synthesis of metal-encased nutraceutical nano-hybrids has been a target for research over the last few years. In the present investigation, we have reported temperature dependent facile synthesis of silver nanoparticles using FDA approved c phycocyanin (cPC). The cPC conjugated silver nanoparticles (AgcPCNPs) were characterized by TEM, Zeta Potential, UV-vis, XPS, FTIR, and CD Spectroscopy. The temperature optimization studies suggested the synthesis of stable AgcPCNPs at 40 °C while at higher temperature system shows aggregated appearance. Molecular docking studies predicted the exclusive interaction of C, D, I, and J chains of cPC with the surface of AgNPs. Moreover, AgcPCNPs significantly (p < 0.1 %) counteract the toxic nature of AgNPs on red blood cell by measuring parameters like total RBC count, % hemolysis, % hematocrit, coagulation time, pH, electrolyte concentrations and degree of blood cell lipid peroxidation by the anti-oxidation mechanism. Skin fibroblast in vitro cell migration result suggeststhat AgcPCNPs enhanced the degree of cell movement towards the wound area. Data obtained collectively demonstrate that AgcPCNPs can be a better agent in the dermal wound healing with reduced toxicity with the bi-phasic advantage of cPC as a wound healer and Ag nano-metal as an anti-bacterial agent.

Substrate specificities of Fusarium biosynthetic enzymes explain the genetic basis of a mixed chemotype producing both deoxynivalenol and nivalenol-type trichothecenes.

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.

Unfolding of CBP21, a lytic polysaccharide monooxygenase, without dissociation of its copper ion cofactor.

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu2+ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, tp , for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of tp in the presence of excess Cu2+ was around 75°C, indicating that Cu2+ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu2+ ⇌ U∙Cu2+ , where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol-1 in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu2+ complex itself.