Six new pyranonaphthoquinone derivatives, gunacin A-E (2-7), along with the known compounds gunacin (1) and the isocoumarin derivative (+) orthosporin (8), were isolated from the fungus Exobasidium sp. Their chemical structures were elucidated by X-ray crystallography, extensive spectroscopic analysis supported by ROESY experiments, and mass spectrometry. Two tested compounds (1, 5) demonstrated high activity against Leishmania mexicana and four salivarian Trypanosoma species, with the lowest detected EC50 value of 0.02-0.24 μM, a value that is comparable to those of currently used drugs. In addition, compounds 1, 3, 5, 6, and 7 demonstrated antibacterial properties at micromolar concentrations, while 1, 5, 6, and 7 exhibited moderate antifungal activity (MIC 33.3-66.7 μM). In cytotoxicity assays, the compounds exhibited a range of toxicity against mammalian Jurkat, RAT2, MDCK cell lines, HeLa cells, and fibroblasts, with inhibition levels varying from strong to minimal inhibition (EC50 = 0.03-125 μM). This study is among the first to explore Exobasidium, a genus of phytopathogenic fungi and highlights the untapped potential of smut fungi (Basidiomycota: Ustilaginomycetes). The discovery of gunacins, which exhibit potent antiprotozoal activity at submicromolar concentrations, suggests a promising avenue for the development of antiprotozoal agents.

Ambrosia fungi colonize dead trees, providing nutrition for ambrosia beetles. A key aspect of this symbiosis is the ability of fungi to colonize and dominate the wood around the beetle tunnels. Hypotheses for these dynamics include active beetle management, fungal inoculation priority, and the chemical ecology of the fungus. The ecological role of allelochemicals produced by ambrosia fungi is unknown. This study presents an analysis of secondary metabolites from Geosmithia eupagioceri (Ascomycota: Hypocreales). Eight extracellular compounds were identified: 5-hydroxymethyl-2-furancarboxylic acid, several hydroxybenzoic acid derivatives, 4-hydroxyphenylacetic acid (4-HPA), 4-HPA methyl ester, tyrosol, and thymine. Most compounds show cross-taxon activity, suppressing the growth of bacteria, fungi, a nematode, and a mite. We also point out that these compounds also have potential to modulate the physiology of their producer, the beetle host and associated microbes through synergism.

This study recognized biologically produced gold nanoparticles (AuNP) as multiple cargo carriers with a perspective of drugs delivery into specialized tumor cells in vivo. Paclitaxel (PTX), transferrin, and antimiR-135b were conjugated with AuNPs and their uptake by mouse tumor cells in an induced breast cancer model was investigated. Each of the above-mentioned molecules was conjugated to the AuNPs separately as well as simultaneously, loading efficiency of each cargo was assessed, and performance of the final product (FP) was assessed. After tumor induction in BALB/c mice, sub-IC50 doses of FP as well as control AuNPs, PTX, and PBS were administered in vivo. Round AuNPs were prepared using Fusarium oxysporum and exhibited size of 13 ± 1.3 nm and zeta potential of -35.8 ± 1.3 mV. The cytotoxicity of individual conjugates and FP were tested by MTT assay in breast tumor cells 4T1 and non-tumor fibroblasts NIH/3T3 cells. The conjugation of the individual molecules with AuNPs was confirmed and FP (size of 54 ± 14 nm and zeta potential of -31.9 ± 2.08 mV) showed higher 4T1-specific toxicity in vitro when compared to control conjugates. After in vivo application of the FP, TEM analyses proved presence of AuNPs in the tumor cells. Hematoxylin and eosin staining of the tumor tissue revealed that the FP group exhibited the highest amounts of inflammatory, necrotic, and apoptotic cells in contrast to control groups. Finally, qPCR results showed that FP could transfect and suppress miR-135b expression in vivo, confirming the tumor-targeting properties of FP. The capacity of biologically produced gold nanoparticles to conjugate with multiple decorative molecules while remaining their stability and effective intracellular uptake makes them promising alternative strategy superior to current drug carriers.

This study recognized biologically produced gold nanoparticles (AuNPs) as multiple cargo carriers with a perspective of drug delivery into specialized tumor cells in vivo. Paclitaxel (PTX), transferrin, and antimiR-135b were conjugated with AuNPs and their uptake by mouse tumor cells in an induced breast cancer model was investigated. Each of the above-mentioned molecules was conjugated to the AuNPs separately as well as simultaneously, loading efficiency of each cargo was assessed, and performance of the final product (FP) was judged. After tumor induction in BALB/c mice, sub-IC50 doses of FP as well as control AuNPs, PTX, and phosphate buffered saline were administered in vivo. Round AuNPs were prepared using Fusarium oxysporum and exhibited a size of 13 +/- 1.3 nm and a zeta potential of -35.8 +/- 1.3 mV. The cytotoxicity of individual conjugates and FP were tested by MTT assay in breast tumor cells 4T1 and nontumor fibroblasts NIH/3T3 cells. The conjugation of individual molecules with AuNPs was confirmed, and FP (size of 54 +/- 14 nm and zeta potential of -31.9 +/- 2.08 mV) showed higher 4T1-specific toxicity in vitro when compared to control conjugates. After in vivo application of the FP, transmission electron microscopy analyses proved the presence of AuNPs in the tumor cells. Hematoxylin and eosin staining of the tumor tissue revealed that the FP group exhibited the highest amounts of inflammatory, necrotic, and apoptotic cells in contrast to the control groups. Finally, qPCR results showed that FP could transfect and suppress miR-135b expression in vivo, confirming the tumor-targeting properties of FP. The capacity of biologically produced gold nanoparticles to conjugate with multiple decorative molecules while retaining their stability and effective intracellular uptake makes them a promising alternative strategy superior to current drug carriers.

Ergot, the genus Claviceps comprises several deeply diverged lineages, recently classified as sections. Among them, the section Pusillae, is the most speciose, with a centre of distribution in Africa but occurring worldwide, often as a consequence of its invasive potential. This section includes the most severe plant pathogens such as Claviceps africana and C. gigantea, responsible for toxicoses and a significant reduction in the seed yields of Sorghum and Zea. In this study we surveyed ergot diversity in South Africa, focusing on grasses native to this region, but known for their high potential of invasiveness. The revision based on molecular and phenotypic markers revealed 16 species, with a high proportion of undescribed diversity, confirming Africa as a hot spot for this section. Five new species, Claviceps tulasnei, Claviceps eulaliae, Claviceps hypertheliae, Claviceps fredericksoniae and Claviceps arundinellae were described from Setaria, Eulalia, Hyperthelia, Miscanthus and Arundinella respectively. Claviceps texensis infecting Cenchrus, previously only identified from the same host in Texas, USA, was confirmed to be present in Africa, which is assumed to be its primary area of distribution. In addition, the host grass genus Anthephora is newly reported as a host of Claviceps digitariae. The most of the taxa were negligible concerning alkaloid production, with the exception of C. fredericksoniae, which is a sister of potent alkaloid producer C. africana, and produces mainly DH-ergosine, together with traces of DH-ergocornine. The host/parasite associations within Pusillae section is very narrow, suggesting that co-speciation is the major speciation driver in this group. Host grasses of the described species are already recognised invasive species and their ovarial parasites need to be monitored. This is highlighted by the fact that all Pusillae produced air-borne secondary conidia, which is autapomorphy of this section and considered to be important for their invasive abilities.

Naphthoquinones isolated from Quambalaria cyanescens (quambalarines) are natural pigments possessing significant cytotoxic and antimicrobial properties. Determining the structure of naphthoquinone compounds is important for the understanding of their biological activities and the informed synthesis of related analogues. Identifying quambalarines is challenging, because they contain a hydroxylated naphthoquinone scaffold and have limited solubility. Here, we report a detailed structural study of quambalarine derivatives, which form strong intramolecular hydrogen bonds (IMHBs) that enable the formation of several tautomers; these tautomers may complicate structural investigation due to their fast interconversion. To investigate tautomeric equilibria and identify new quambalarines, we complemented the experimental NMR spectroscopy data with density functional theory (DFT) calculations.

Aspergillus section Nidulantes encompasses almost 80 homothallic and anamorphic species, mostly isolated from soil, plant material, or the indoor environment. Some species are clinically relevant or produce mycotoxins. This study reevaluated the species boundaries within several clades of section Nidulantes. Five data sets were assembled, each containing presumptive new species and their closest relatives, and phylogenetic and phenotypic analyses were performed. We tested the hypotheses that the newly isolated or reexamined strains constitute separate species (splitting approach) or should be treated as part of broadly defined species (lumping approach). Four DNA sequence loci were amplified, internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA and partial sequences of the β-tubulin (benA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes. The latter three loci were used for the phylogenetic analysis and served as input for single-locus (GMYC, bGMYC, PTP, and bPTP) and multilocus (STACEY and BP&P) species delimitation analyses. The phenotypic analysis comprised macro- and micromorphology (including scanning electron microscopy) and comparison of cardinal growth temperatures. The phylogenetic analysis supported the splitting hypothesis in all cases, and based on the combined approach, we propose six new species, four that are homothallic and two anamorphic. Four new species were isolated from the indoor environment (Jamaica, Trinidad and Tobago, USA), one originated from soil (Australia), and one from a kangaroo rat cheek pouch (USA).

Ergot, fungal genus Claviceps, are worldwide distributed grass pathogens known for their production of toxic ergot alkaloids (EAs) and the great agricultural impact they have on both cereal crop and farm animal production. EAs are traditionally considered as the only factor responsible for ergot toxicity. Using broad sampling covering 13 ergot species infecting wild or agricultural grasses (including cereals) across Europe, USA, New Zealand, and South Africa we showed that the content of ergochrome pigments were comparable to the content of EAs in sclerotia. While secalonic acids A-C (SAs), the main ergot ergochromes (ECs), are well known toxins, our study is the first to address the question about their contribution to overall ergot toxicity. Based on our and published data, the importance of SAs in acute intoxication seems to be negligible, but the effect of chronic exposure needs to be evaluated. Nevertheless, they have biological activities at doses corresponding to quantities found in natural conditions. Our study highlights the need for a re-evaluation of ergot toxicity mechanisms and further studies of SAs' impact on livestock production and food safety.

The ergot, genus Claviceps, comprises approximately 60 species of specialised ovarial grass parasites famous for the production of food toxins and pharmaceutics. Although the ergot has been known for centuries, its evolution have not been resolved yet. Our approach combining multilocus phylogeny, molecular dating and the study of ecological, morphological and metabolic features shows that Claviceps originated in South America in the Palaeocene on a common ancestor of BEP (subfamilies Bambusoideae, Ehrhartoideae, Pooideae) and PACMAD (subfamilies Panicoideae, Aristidoideae, Chloridoideae, Micrairoideae, Arundinoideae, Danthonioideae) grasses. Four clades described here as sections diverged during the Paleocene and Eocene. Since Claviceps are parasitic fungi with a close relationship with their host plants, their evolution is influenced by interactions with the new hosts, either by the spread to a new continent or the radiation of the host plants. Three of the sections possess very narrow host ranges and biogeographical distributions and have relatively low toxicity. On the contrary, the section Claviceps, comprising the rye ergot, C. purpurea, is unique in all aspects. Fungi in this section of North American origin have spread all over the world and infect grasses in all subfamilies as well as sedges, and it is the only section synthesising toxic ergopeptines and secalonic acids. The evolutionary success of the Claviceps section members can be explained by high toxin presence, serving as feeding deterrents and playing a role in their protective mutualism with host plants. Closely related taxa Neoclaviceps monostipa and Cepsiclava phalaridis were combined into the genus Aciculosporium.

Abnormalities in cancer metabolism represent potential targets for cancer therapy. We have recently identified a natural compound Quambalarine B (QB), which inhibits proliferation of several leukemic cell lines followed by cell death. We have predicted ubiquinone binding sites of mitochondrial respiratory complexes as potential molecular targets of QB in leukemia cells. Hence, we tracked the effect of QB on leukemia metabolism by applying several omics and biochemical techniques. We have confirmed the inhibition of respiratory complexes by QB and found an increase in the intracellular AMP levels together with respiratory substrates. Inhibition of mitochondrial respiration by QB triggered reprogramming of leukemic cell metabolism involving disproportions in glycolytic flux, inhibition of proteins O-glycosylation, stimulation of glycine synthesis pathway, and pyruvate kinase activity, followed by an increase in pyruvate and a decrease in lactate levels. Inhibition of mitochondrial complex I by QB suppressed folate metabolism as determined by a decrease in formate production. We have also observed an increase in cellular levels of several amino acids except for aspartate, indicating the dependence of Jurkat (T-ALL) cells on aspartate synthesis. These results indicate blockade of mitochondrial complex I and II activity by QB and reduction in aspartate and folate metabolism as therapeutic targets in T-ALL cells. Anti-cancer activity of QB was also confirmed during in vivo studies, suggesting the therapeutic potential of this natural compound.

Results of a survey and study of the Claviceps purpurea group of species in South Africa are being presented and five new species are described. Morphological descriptions are based on the anamorphs and four nuclear genetic loci. Claviceps fimbristylidis sp. nov. on Fimbristylis complanata was discovered wide-spread across five provinces of the country associated with water and represents the fourth Claviceps species recorded from the Cyperaceae. Claviceps monticola sp. nov. is described from Brachypodium flexum growing in mountain forests in Mpumalanga Province, as well as the northern Drakensberg southwards into the Eastern Cape Province. Claviceps pazoutovae sp. nov. is recorded from Stipa dregeana var. dregeana and Ehrharta erecta var. erecta, also associated with these mountain ranges. Claviceps macroura sp. nov. is recorded from Cenchrus macrourus from the Eastern Cape and Claviceps capensis sp. nov. from Ehrharta villosa var. villosa is recorded from the Western Cape Province. Claviceps cyperi, only recorded from South Africa is included in the study. Ergot alkaloid profiles of all species are provided and showed similarity to C. purpurea. Only C. cyperi and in lesser degree C. capensis, C. macroura, and C. pazoutovae produced ergot alkaloids in clinically significant amounts. Several reported species infect invasive grass species, native to South Africa, and thus represent potentially invasive species.

Quambalarine B (QB) is a secondary metabolite produced by the basidiomycete Quambalaria cyanescens with potential anticancer activity. Here we report that QB at low micromolar concentration inhibits proliferation of several model leukemic cell lines (Jurkat, NALM6, and REH), whereas higher concentrations induce cell death. By contrast, the effect of QB on primary leukocytes (peripheral blood mononuclear cells) is significantly milder with lower toxicity and cytostatic activity. Moreover, QB inhibited expression of the C-MYC oncoprotein and mRNA expression of its target genes, LDHA, PKM2, and GLS. Finally, QB blocked the phosphorylation of P70S6K, a downstream effector kinase in mTOR signaling that regulates translation of C-MYC. This observation could explain the molecular mechanism behind the antiproliferative and cytotoxic effects of QB on leukemic cells. Altogether, our results establish QB as a promising molecule in anticancer treatment.

Pathogenic and non-pathogenic related microorganisms differ in secondary metabolite production. Here we show that riboflavin overproduction by a fungal pathogen and its hyperaccumulation in affected host tissue exacerbates a skin infection to necrosis. In white-nose syndrome (WNS) skin lesions caused by Pseudogymnoascus destructans, maximum riboflavin concentrations reached up to 815 μg ml−1, indicating bioaccumulation and lack of excretion. We found that high riboflavin concentrations are cytotoxic under conditions specific for hibernation, affect bats’ primary fibroblasts and induce cell detachment, loss of mitochondrial membrane potential, polymerization of cortical actin, and cell necrosis. Our results explain molecular pathology of WNS, where a skin infection becomes fatal. Hyperaccumulation of vitamin B2 coupled with reduced metabolism and low tissue oxygen saturation during hibernation prevents removal of excess riboflavin in infected bats. Upon reperfusion, oxygen reacts with riboflavin resulting in dramatic pathology after arousal. While multiple molecules enable invasive infection, riboflavin-associated extensive necrosis likely contributes to pathophysiology and altered arousal pattern in infected bats. Bioaccumulation of a vitamin under natural infection represents a novel condition in a complex host-pathogen interplay.

Tyrosine kinases (TKs) play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015) [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015) [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002065.

Four strains of the fungus Quambalaria cyanescens (Basidiomycota: Microstromatales), were used for the determination of secondary metabolites production and their antimicrobial and biological activities. A new naphthoquinone named quambalarine A, (S)-(+)-3-(5-ethyl-tetrahydrofuran-2-yliden)-5,7,8-trihydroxy-2-oxo-1,4-naphthoquinone (1), together with two known naphthoquinones, 3-hexanoyl-2,5,7,8-tetrahydroxy-1,4-naphthoquinone (named here as quambalarine B, 2) and mompain, 2,5,7,8-tetrahydroxy-1,4-naphthoquinone (3) were isolated. Their structures were determined by single-crystal X-ray diffraction crystallography, NMR and MS spectrometry. Quambalarine A (1) had a broad antifungal and antibacterial activity and is able inhibit growth of human pathogenic fungus Aspergillus fumigatus and fungi co-occurring with Q. cyanescens in bark beetle galleries including insect pathogenic species Beauveria bassiana. Quambalarine B (2) was active against several fungi and mompain mainly against bacteria. The biological activity against human-derived cell lines was selective towards mitochondria (2 and 3); after long-term incubation with 2, mitochondria were undetectable using a mitochondrial probe. A similar effect on mitochondria was observed also for environmental competitors of Q. cyanescens from the genus Geosmithia.

A new, efficient, and general semisynthesis of hydnocarpin-type flavonolignans was developed and optimized, enabling gram-scale production of hydnocarpin D (2). Moreover, the syntheses of optically pure hydnocarpin isomers [(10R,11R)-hydnocarpin (1a), (10R,11R)-hydnocarpin D (2a), and (10S,11S)-hydnocarpin D (2b)], as well as the synthesis of isohydnocarpin (8), were achieved for the first time utilizing this new method. The synthesis is based on the two-step transformation of the readily available flavonolignans from milk thistle (Silybum marianum), accessible by isolation from the commercial extract silymarin. The first step relies on the regioselective formylation of the C-3 hydroxy group of the dihydroflavonol-type precursor using the Vilsmeier-Haack reagent, followed by formic acid elimination by triethylamine in the second step. The synthesized compounds were effective inhibitors of Staphylococcus aureus biofilm formation, with (10S,11S)-hydnocarpin D (2b) being the most potent inhibitor. Furthermore, the effect of glucose on biofilm formation was tested, and glucose decreased the biofilm inhibitory activity of 2b. Moreover, 2b increased the susceptibility of Staph. aureus to enrofloxacin.

A strain of Biatriospora sp. CCF 4378 was tested for the production of secondary metabolites under submerged fermentation conditions. Eleven compounds were isolated from the culture broth, and the structures of these compounds were determined using HRMS, NMR and X-ray analysis. In addition to six known naphthoquinone derivatives, i.e. ascomycone A, ascomycone B, 6-deoxyfusarubine, 6-deoxyanhydrofusarubine, herbarine and balticol A, one derivative of 2-azaanthraquinone, 6-deoxybostrycoidine, was also identified. Four new natural pyranonaphthoquinones were found, and these natural products were pleorubrin A, pleorubrin B, pleorubrin C and pleorubrin D. The toxicity on human cell lines of the crude naphthoquinone fraction and pure 6-deoxybostrycoidin, ascomycone B, pleorubrin B and 6-deoxyfusarubin was tested. Ascomycone B and 6-deoxyfusarubin elicited rapid cytotoxicity at micromolar concentrations.

Silent information regulators are NAD+-dependent enzymes that display differential specificity toward acetylated substrates. This report provides first evidence for deacetylation activity of CobB1 in Streptomyces coelicolor. The protein is highly conserved in streptomycetes. The CobB1 protein catalytically removes the acetyl group from acetylated bovine serum albumin. In the absence of NAD+ or when NAD+ was substituted with nicotinamide, deacetylation was stopped. We isolated gene encoding AcetylCoA synthetaseA. The recombinant enzyme produces Acetyl-CoA from acetate. The highest acsA-mRNA level was detected in cells from the exponential phase of growth, and then decreased in transition and stationary phases of growth. Acetylated acsA loses the ability to transfer acetate to CoA. Deacetylation of the enzyme required CobB1, ATP-Mg2, and NAD+. Using specific antibodies against acetylated lys, CobB1, and acsA, we found relationship between level of CobB1 and acetylation of acsA, indicating that CobB1 is involved in regulating the acetylation level of acsA and consequently its activity. It was found that 1-acetyl-tetrahydroxy and 1-acetyl pentahydroxy antraquinone inhibit the deacetylation activity of CobB1.

A new polyene macrolide family, closely related to the pentaene macrolide antibiotic roflamycoin, was isolated from the both fermentation broth and biomass of Streptomyces durmitorensis wild-type strain MS405. The main compound was identified by NMR and Fourier transform ion cyclotron resonance mass spectrometry as 32,33-didehydroroflamycoin (1; DDHR). Additional four structurally related compounds were determined solely by MS analysis. DDHR induces cell death by apoptosis in various cancer cell lines as demonstrated by DNA fragmentation. Striking feature of DDHR is its internal fluorescence allowing visualization of labeled plasma membranes and internal membrane structures.