Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro

Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT.

Attempting to rewrite History:Challenges with the analysis of histidine-phosphorylated peptides

A significant number of proteins in both eukaryotes and prokaryotes are known to be post-translationally modified by the addition of phosphate, serving as a means of rapidly regulating protein function. Phosphorylation of the amino acids serine, threonine and tyrosine are the focus of the vast majority of studies aimed at elucidating the extent and roles of such modification, yet other amino acids, including histidine and aspartate, are also phosphorylated. Although histidine phosphorylation is known to play extensive roles in signalling in eukaryotes, plants and fungi, roles for phosphohistidine are poorly defined in higher eukaryotes. Characterization of histidine phosphorylation aimed at elucidating such information is problematic due to the acid-labile nature of the phosphoramidate bond, essential for many of its biological functions. Although MSbased strategies have proven extremely useful in the analysis of other types of phosphorylated peptides, the chromatographic procedures essential for such approaches promote rapid hydrolysis of phosphohistidinecontaining peptides. Phosphate transfer to non-biologically relevant aspartate residues during MS analysis further complicates the scenario. © 2013 Biochemical Society.

Chaotropic and hydrophobic stress mechanisms of antifungal substances

Growth and metabolism of fungi can be curtailed by chaotropic solutes and hydrophobic substances, both of which can weaken or inhibit non-covalent interactions within and between macromolecular systems. Here we explore the potential to utilize the fungistatic and fungicidal activities of such stressors as the basis for commercial formulations. A method was developed for the quantification of chaotropicity, which can be used for chemically diverse substances, in order elucidate roles of chaotropicity and hydrophobicity in microbial ecology (both of which are sufficiently potent to limit the Earth’s microbial biosphere). A large number of naturally occurring substances act as chaotropic or hydrophobic stressors including aliphatic alcohols, salts such as MgCl2, aromatics such as phenol, and hydrocarbons such as hexane and octene. We suggest that these stress parameters provide the (hitherto unidentified) modes-of-action for some extant antifungal products. The findings are discussed in relation to the development of a new generation of antifungals.

Determinants of root-associated fungal communities within Asteraceae in a semi-arid grassland

Summary

1.While plant–fungal interactions are important determinants of plant community assembly and ecosystem functioning, the processes underlying fungal community composition are poorly understood.
2.Here, we studied for the first time the root-associated eumycotan communities in a set of co-occurring plant species of varying relatedness in a species-rich, semi-arid grassland in Germany. The study system provides an opportunity to evaluate the importance of host plants and gradients in soil type and landscape structure as drivers of fungal community structure on a relevant spatial scale. We used 454 pyrosequencing of the fungal internal transcribed spacer region to analyse root-associated eumycotan communities of 25 species within the Asteraceae, which were sampled at different locations within a soil type gradient. We partitioned the variance accounted for by three predictors (host plant phylogeny, spatial distribution and soil type) to quantify their relative roles in determining fungal community composition and used null model analyses to determine whether community composition was influenced by biotic interactions among the fungi.
3.We found a high fungal diversity (156 816 sequences clustered in 1100 operational taxonomic units (OTUs)). Most OTUs belonged to the phylum Ascomycota (35.8%); the most abundant phylotype best-matched Phialophora mustea. Basidiomycota were represented by 18.3%, with Sebacina as most abundant genus. The three predictors explained 30% of variation in the community structure of root-associated fungi, with host plant phylogeny being the most important variance component. Null model analysis suggested that many fungal taxa co-occurred less often than expected by chance, which demonstrates spatial segregation and indicates that negative interactions may prevail in the assembly of fungal communities.
4.Synthesis. The results show that the phylogenetic relationship of host plants is the most important predictor of root-associated fungal community assembly, indicating that fungal colonization of host plants might be facilitated by certain plant traits that may be shared among closely related plant species.

Choosing and using diversity indices: Insights for ecological applications from the German Biodiversity Exploratories

Biodiversity, a multidimensional property of natural systems, is difficult to quantify partly because of the multitude of indices proposed for this purpose. Indices aim to describe general properties of communities that allow us to compare different regions, taxa, and trophic levels. Therefore, they are of fundamental importance for environmental monitoring and conservation, although there is no consensus about which indices are more appropriate and informative. We tested several common diversity indices in a range of simple to complex statistical analyses in order to determine whether some were better suited for certain analyses than others. We used data collected around the focal plant Plantago lanceolata on 60 temperate grassland plots embedded in an agricultural landscape to explore relationships between the common diversity indices of species richness (S), Shannon's diversity (H'), Simpson's diversity (D1), Simpson's dominance (D2), Simpson's evenness (E), and Berger–Parker dominance (BP). We calculated each of these indices for herbaceous plants, arbuscular mycorrhizal fungi, aboveground arthropods, belowground insect larvae, and P. lanceolata molecular and chemical diversity. Including these trait-based measures of diversity allowed us to test whether or not they behaved similarly to the better studied species diversity. We used path analysis to determine whether compound indices detected more relationships between diversities of different organisms and traits than more basic indices. In the path models, more paths were significant when using H', even though all models except that with E were equally reliable. This demonstrates that while common diversity indices may appear interchangeable in simple analyses, when considering complex interactions, the choice of index can profoundly alter the interpretation of results. Data mining in order to identify the index producing the most significant results should be avoided, but simultaneously considering analyses using multiple indices can provide greater insight into the interactions in a system.

Arbuscular mycorrhizal fungal communities are phylogenetically clustered at small scales

Next-generation sequencing technologies with markers covering the full Glomeromycota phylum were used to uncover phylogenetic community structure of arbuscular mycorrhizal fungi (AMF) associated with Festuca brevipila. The study system was a semi-arid grassland with high plant diversity and a steep environmental gradient in pH, C, N, P and soil water content. The AMF community in roots and rhizosphere soil were analyzed separately and consisted of 74 distinct operational taxonomic units (OTUs) in total. Community-level variance partitioning showed that the role of environmental factors in determining AM species composition was marginal when controlling for spatial autocorrelation at multiple scales. Instead, phylogenetic distance and spatial distance were major correlates of AMF communities: OTUs that were more closely related (and which therefore may have similar traits) were more likely to co-occur. This pattern was insensitive to phylogenetic sampling breadth. Given the minor effects of the environment, we propose that at small scales closely related AMF positively associate through biotic factors such as plant-AMF filtering and interactions within the soil biota.

Diversity and abundance of soil arthropods in urban and suburban holm oak stands

We investigated the soil arthropod communities of urban and suburban holm oak (Quercus ilex L.) stands in a small (Siena) and a large Italian city (Naples) and tested whether the abundance and diversity of higher arthropod taxa are affected by the biotic and abiotic conditions of urban forest soils, including pollution. Acarina and Collembola were the dominant taxa in both cities. In Siena the total number of arthropod individuals collected in the samples was over 1/3 greater than in Naples, but all diversity indices scored higher in Naples than in Siena, probably in response to the higher heterogeneity of microclimatic and pedological conditions found in Naples study area. Oribatids resulted twice more abundant in Siena and so were the total mites with respect to Collembola. While “taxonomic richness” per site increased with distance from road traffic, entropy and evenness indices scored higher at the two ends of the impact gradient in both cities. The overall variation in basic pedological and microbiological soil parameters positively correlated with the total abundance of arthropods, and negatively correlated with their taxonomic richness. At the resolution employed, no significant relation emerged between anthropogenic factors, such as traffic load and soil pollution, and the arthropod fauna density and variety. These results are consistent with conclusions drawn from a previous study on the enchytraeid fauna examined at species level, which is remarkable considering the different taxonomic resolutions of the two studies. CCA results suggest that the higher abundance of Oribatid mites, Protura and Thysanura and the lower abundance of Diplopoda and Symphyla in Siena could depend on a higher fungi/bacteria ratio. This observation can be interpreted in terms of differences in fungi and bacteria between the two cities: Siena is shifted towards the fungal decomposition channel, which supports taxa such as oribatid mites, while Naples is shifted towards the bacterial channel, which supports chiefly detritivorous groups, such as diplopods.

Accuracy of LightCycler SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review and meta-analysis

Purpose: There is an urgent need to develop diagnostic tests to improve the detection of pathogens causing life-threatening infection (sepsis). SeptiFast is a CE-marked multi-pathogen real-time PCR system capable of detecting DNA sequences of bacteria and fungi present in blood samples within a few hours. We report here a systematic review and meta-analysis of diagnostic accuracy studies of SeptiFast in the setting of suspected sepsis.

Methods: A comprehensive search strategy was developed to identify studies that compared SeptiFast with blood culture in suspected sepsis. Methodological quality was assessed using QUADAS. Heterogeneity of studies was investigated using a coupled forest plot of sensitivity and specificity and a scatter plot in receiver operator characteristic space. Bivariate model method was used to estimate summary sensitivity and specificity.

Results: From 41 phase III diagnostic accuracy studies, summary sensitivity and specificity for SeptiFast compared with blood culture were 0.68 (95 % CI 0.63–0.73) and 0.86 (95 % CI 0.84–0.89) respectively. Study quality was judged to be variable with important deficiencies overall in design and reporting that could impact on derived diagnostic accuracy metrics.

Conclusions: SeptiFast appears to have higher specificity than sensitivity, but deficiencies in study quality are likely to render this body of work unreliable. Based on the evidence presented here, it remains difficult to make firm recommendations about the likely clinical utility of SeptiFast in the setting of suspected sepsis.

Water and temperature relations of soil Actinobacteria

Actinobacteria perform essential functions within soils, and are dependent on available water to do so. We determined the water-activity (a_w) limits for cell division of Streptomyces albidoflavus, Streptomyces rectiviolaceus, Micromonospora grisea and Micromonospora (JCM 3050) over a range of temperatures, using culture media supplemented with a biologically permissive solute (glycerol). Each species grew optimally at 0.998 a_w (control; no added glycerol) and growth rates were near-optimal in the range 0.971–0.974 (1 M glycerol) at permissive temperatures. Each was capable of cell division at 0.916–0.924 a_w (2 M glycerol), but only S. albidoflavus grew at 0.895 or 0.897 a_w (3 M glycerol, at 30 and 37°C respectively). For S. albidoflavus, however, no growth occurred on media at ≤ 0.870 (4 M glycerol) during the 40-day assessment period, regardless of temperature, and a theoretical limit of 0.877 a_w was derived by extrapolation of growth curves. This level of solute tolerance is high for non-halophilic bacteria, but is consistent with reported limits for the growth and metabolic activities of soil microbes. The limit, within the range 0.895–0.870 a_w, is very much inferior to those for obligately halophilic bacteria and extremely halophilic or xerophilic fungi, and is inconsistent with earlier reports of cell division at 0.500 a_w. These findings are discussed in relation to planetary protection policy for space exploration and the microbiology of arid soils.

Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (a_w) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 a_w. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 a_w). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 a_w for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (~0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 a_w for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

A rapid HPLC protocol for detection of polyols and trehalose

A procedure was developed to extract polyols and trehalose (protectants against stress) from fungal conidia. Conidia were sonicated (120 s) and immersed in a boiling water bath (5.5 min) to optimize extraction of polyols and trehalose, respectively. A rapid method was developed to separate and detect low-molecular-weight polyols and trehalose using high-performance liquid chromatography (HPLC). An ion exchange column designed for standard carbohydrate analysis was used in preference to one designed for sugar alcohol separation. This resulted in rapid elution (less than 5 min), without sacrificing peak resolution. The use of a pulsed electrochemical detector (gold electrode) resulted in limits of reliable quantification as low as 1.6 μg ml-1 for polyols and 2.8 μg ml-1 for trehalose. This is very sensitive and rapid method by which these protectants can be analysed. It avoids polyol derivatization that characterizes analysis by gas chromatography and the long run times (up to 45 min) that typify HPLC analysis using sugar alcohol columns.

Culture Age, temperature, and pH affect the polyol and trehalose contents of fungal propagules

The growth and conidial physiology of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus were studied under different conditions. The effects of culture age (up to 120 days), temperature (5 to 35°C), and pH (2.9 to 11.1) were determined. Growth was optimal at pH 5 to 8 for each isolate and between 20 and 35°C, depending on the isolate. The predominant polyol in conidia was mannitol, with up to 39, 134, and 61 mg g of conidia-1 for B. bassiana, M. anisopliae, and P. farinosus, respectively. Conidia of M. anisopliae contained relatively small amounts of lower-molecular-weight polyols and trehalose (less than 25 mg g-1 in total) in all treatments. Conidia of B. bassiana and P. farinosus contained up to 30, 32, and 25 mg of glycerol, erythritol, and trehalose, respectively, g-1, depending on the treatment. Conidia of P. farinosus contained unusually high amounts of glycerol and erythritol at pH 2.9. The apparent effect of pH on gene expression is discussed in relation to the induction of a water stress response. To our knowledge, this is the first report of polyols and trehalose in fungal propagules produced over a range of temperature or pH. Some conditions and harvesting times were associated with an apparent inhibition of synthesis or accumulation of polyols and trehalose. This shows that culture age and environmental conditions affect the physiological quality of inoculum and can thereby determine its potential for biocontrol.

Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability

The insect pathogen Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosos can be effective biocontrol agents when relative humidity (RH) is close to 100%. At reduced water availability, germination of propagules, and therefore host infection, cannot occur. Cultures of B. bassiana, M. anisopliae and P. farinosus were grown under different conditions to obtain conidia with a modified polyol and trehalose content. Conidia with higher intracellular concentrations of glycerol and erythritol germinated both more quickly and at lower water activity (a(w)) than those from other treatments. In contrast, conidia containing up to 235.7 mg trehalose g-1 germinated significantly (P < 0 05) more slowly than those with an equivalent polyol content but less trehalose, regardless of water availability. Conidia from control treatments did not germinate below 0.951 - 0.935 a(w) (≡ 95.1 - 93.5% RH). In contrast, conidia containing up to 164.6 mg glycerol plus erythritol g-1 germinated down to 0.887 a(w) (≡ 88.7% RH). These conidia germinated below the water availability at which mycelial growth ceases (0.930 - 0.920 a(w)). Germ tube extension rates reflected the percentage germination of conidia, so the most rapid germ tube growth occurred after treatments which produced conidia containing the most glycerol and erythritol. This study shows for the first time that manipulating polyol content can extend the range of water availability over which fungal propagules can germinate. Physiological manipulation of conidia may improve biological control of insect pests in the field.

Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: Potent activity against oral pathogens

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50. μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10. μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

Biotransformation of zearalenone and zearalenols to their major glucuronide metabolites reduces estrogenic activity

Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungi. Once ingested, ZEN may be absorbed andmetabolised to a- and b-zearalenol (a-ZOL, b-ZOL), and to a lesser extent a- and b-zearalanol (a-ZAL,b-ZAL). Further biotransformation to glucuronide conjugates also occurs to facilitate the elimination ofthese toxins from the body. Unlike ZEN and its metabolites, information regarding the estrogenic activityof these glucuronide conjugates in various tissues is lacking. ZEN-14-O-glucuronide, a-ZOL-14-O-glucuronide,a-ZOL-7-O-glucuronide, b-ZOL-14-O-glucuronide and b-ZOL-16-O-glucuronide, previouslyobtained as the major products from preparative enzymatic synthesis, were investigated for their potentialto cause endocrine disruption through interference with estrogen receptor transcriptional activity.All five glucuronide conjugates showed a very weak agonist response in an estrogen responsive reportergene assay (RGA), with activity ranging from 0.0001% to 0.01% of that of 17b-estradiol, and also lessthan that of ZEN, a-ZOL and b-ZOL which have previously shown estrogenic potencies of the order 17bestradiol> a-ZOL > ZEN > b-ZOL. Confirmatory mass spectrometry revealed that any activity observedwas likely a result of minor deconjugation of the glucuronide moiety. This study confirms that formationof ZEN and ZOL glucuronides is a detoxification reaction with regard to estrogenicity, serving as a potentialhost defence mechanism against ZEN-induced estrogenic activity.