Global patterns in endemicity and vulnerability of soil fungi.

Fungi are highly diverse organisms, which provide multiple ecosystem services. However, compared with charismatic animals and plants, the distribution patterns and conservation needs of fungi have been little explored. Here, we examined endemicity patterns, global change vulnerability and conservation priority areas for functional groups of soil fungi based on six global surveys using a high-resolution, long-read metabarcoding approach. We found that the endemicity of all fungi and most functional groups peaks in tropical habitats, including Amazonia, Yucatan, West-Central Africa, Sri Lanka, and New Caledonia, with a negligible island effect compared with plants and animals. We also found that fungi are predominantly vulnerable to drought, heat and land-cover change, particularly in dry tropical regions with high human population density. Fungal conservation areas of highest priority include herbaceous wetlands, tropical forests, and woodlands. We stress that more attention should be focused on the conservation of fungi, especially root symbiotic arbuscular mycorrhizal and ectomycorrhizal fungi in tropical regions as well as unicellular early-diverging groups and macrofungi in general. Given the low overlap between the endemicity of fungi and macroorganisms, but high conservation needs in both groups, detailed analyses on distribution and conservation requirements are warranted for other microorganisms and soil organisms.

Ethnobiological notes and volatile profiles of two rare Chinese desert truffles.

The production of a distinct profile of volatile organic compounds plays a crucial role in the ecology of hypogeous Ascomycetes, and is also key to their gastronomic relevance. In this study, we explored the aroma components of two rarely investigated Chinese desert truffles, namely Mattirolomyces terfezioides and Choiromyces cerebriformis, using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Our investigation revealed the significant presence of sulphur-containing volatiles in the aroma of M. terfezioides but not in C. cerebriformis. We discussed available information on the distribution of these interesting truffles in China and their use as choice food by local people.

Multitalented Synthetic Antimicrobial Peptides and Their Antibacterial, Antifungal and Antiviral Mechanisms.

Despite the great strides in healthcare during the last century, some challenges still remained unanswered. The development of multi-drug resistant bacteria, the alarming growth of fungal infections, the emerging/re-emerging of viral diseases are yet a worldwide threat. Since the discovery of natural antimicrobial peptides able to broadly hit several pathogens, peptide-based therapeutics have been under the lenses of the researchers. This review aims to focus on synthetic peptides and elucidate their multifaceted mechanisms of action as antiviral, antibacterial and antifungal agents. Antimicrobial peptides generally affect highly preserved structures, e.g., the phospholipid membrane via pore formation or other constitutive targets like peptidoglycans in Gram-negative and Gram-positive bacteria, and glucan in the fungal cell wall. Additionally, some peptides are particularly active on biofilm destabilizing the microbial communities. They can also act intracellularly, e.g., on protein biosynthesis or DNA replication. Their intracellular properties are extended upon viral infection since peptides can influence several steps along the virus life cycle starting from viral receptor-cell interaction to the budding. Besides their mode of action, improvements in manufacturing to increase their half-life and performances are also taken into consideration together with advantages and impairments in the clinical usage. Thus far, the progress of new synthetic peptide-based approaches is making them a promising tool to counteract emerging infections.

Morpho-anatomical and molecular characterization of a native mycorrhizal Amanita species associated with Guapira opposita (Nyctaginaceae) in the brazilian Atlantic Forest.

In this work, we characterize naturally occurring mycorrhizae formed by Amanita viscidolutea on Guapira opposita in the Atlantic Forest in Brazil. We sequenced the rDNA ITS region from the mycorrhizae and basidiomata to identify both symbionts. Amanita viscidolutea mycorrhizae were up to 43 mm long, mostly simple, and unbranched to irregularly pinnate. The fungal mantle surface was velvety to slightly cottony and white to yellowish with silver patches. Hyphal strands were infrequently present. Although the fungal mantle consisted of clampless hyphae, emanating hyphae and hyphal strands had sparsely distributed clamp connections. A unique character of the mycorrhizae was the absence of a Hartig net.

Cytinus under the Microscope: Disclosing the Secrets of a Parasitic Plant.

Well over 1% of all flowering plants are parasites, obtaining all or part of the nutrients they need from other plants. Among this extremely heterogeneous assemblage, the Cytinaceae form a small group of holoparasites, with Cytinus as the main representative genus. Despite the small number of known species and the fact that it doesn't attack crops or plants of economic importance, Cytinus is paradigmatic among parasitic plants. Recent research has indeed disclosed many aspects of host-parasite interactions and reproductive biology, the latter displaying a vast array of adaptive traits to lure a range of animal pollinators. Furthermore, analysis of biological activities of extracts of the most common species of Cytinus has provided evidence that this plant could be a valuable source of compounds with high potential in key applicative areas, namely food production (nutraceuticals) and the development of antimicrobial therapeutics. This article offers a complete overview of our current knowledge of Cytinus.

The Anti-Microbial Peptide (Lin-SB056-1)2-K Reduces Pro-Inflammatory Cytokine Release through Interaction with Pseudomonas aeruginosa Lipopolysaccharide.

The ability of many anti-microbial peptides (AMPs) to modulate the host immune response has highlighted their possible therapeutic use to reduce uncontrolled inflammation during chronic infections. In the present study, we examined the anti-inflammatory potential of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K, which were previously found to have anti-microbial activity against Pseudomonas aeruginosa in in vivo-like models mimicking the challenging environment of chronically infected lungs (i.e., artificial sputum medium and 3-D lung mucosa model). The dendrimeric derivative exerted a stronger anti-inflammatory activity than its monomeric counterpart towards lung epithelial- and macrophage-cell lines stimulated with P. aeruginosa lipopolysaccharide (LPS), based on a marked decrease (up to 80%) in the LPS-induced production of different pro-inflammatory cytokines (i.e., IL-1ß, IL-6 and IL-8). Accordingly, (lin-SB056-1)2-K exhibited a stronger LPS-binding affinity than its monomeric counterpart, thereby suggesting a role of peptide/LPS neutralizing interactions in the observed anti-inflammatory effect. Along with the anti-bacterial and anti-biofilm properties, the anti-inflammatory activity of (lin-SB056-1)2-K broadens its therapeutic potential in the context of chronic (biofilm-associated) infections.

Tannin profile, antioxidant properties, and antimicrobial activity of extracts from two Mediterranean species of parasitic plant Cytinus.

BACKGROUND: Cytinus is small genus of endophytic parasitic plants distributed in South Africa, Madagascar, and in the Mediterranean region. In the latter area, two species occur, Cytinus hypocistis and C. ruber, distinguished by both morphological characters and ecological traits. We characterized the ethanolic and aqueous extracts obtained from the inflorescences of C. hypocistis and C. ruber collected in Sardinia, Italy, and explored their tannin content, antioxidant properties and antimicrobial activities. METHODS: Total phenolic contents were determined by Folin-Ciocalteu spectrophotometric method. Tannin content was determined by HPLC. Antioxidant activity of the extracts was tested with both electron transfer-based (FRAP, TEAC, DPPH) and spectrophotometric HAT methods (ORAC-PYR). The antimicrobial activities of extracts/compounds were evaluated using the broth microdilution method. The bactericidal activity was evaluated using the time-kill method. Biofilm formation was evaluated by crystal violet (CV) staining assay. RESULTS: Characterization of the tannin profile of C. hypocistis and C. ruber revealed a significant amount of gallotannins, in particular 1-O-galloyl-ß-D-glucose. In addition, pentagalloyl-O-ß-D-glucose was present in all extracts, reaching the concentration of 0.117 g/kg in the ethanolic extract of C. hypocistis. C. hypocistis extracts displayed a strongest antioxidant activity than C. ruber extracts. Three Gram-positive bacterial species tested (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium) resulted sensitive to both Cytinus extracts, with MICs ranging from 125 to 500 µg/ml for aqueous extracts and from 31.25 to 250 µg/ml for ethanolic extracts; on the contrary, Gram-negative strains (Pseudomonas aeruginosa and Klebsiella pneumoniae) were not affected by Cytinus extracts. Intriguingly, we observed the suppressive activity of ethanolic extracts of C. hypocistis and C. ruber on biofilm formation of S. epidermidis. Experiments performed with synthetic compounds indicated that pentagalloyl-O-ß-D-glucose is likely to be one of the active antimicrobial components of Cytinus extracts. CONCLUSIONS: These findings show that Cytinus extracts have antimicrobial and antioxidant activities, suggesting a possible application of Cytinus as sources of natural antimicrobials and antioxidants.

The Antimicrobial Peptide lin-SB056-1 and Its Dendrimeric Derivative Prevent Pseudomonas aeruginosa Biofilm Formation in Physiologically Relevant Models of Chronic Infections.

Antimicrobial peptides (AMPs) are promising templates for the development of novel antibiofilm drugs. Despite the large number of studies on screening and optimization of AMPs, only a few of these evaluated the antibiofilm activity in physiologically relevant model systems. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predict the clinical potential of novel AMP-based antimicrobials. In the present study, we examined the antibiofilm activity of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K against Pseudomonas aeruginosa in an in vivo-like three-dimensional (3-D) lung epithelial cell model and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels). Although moderately active when tested alone, lin-SB056-1 was effective in reducing P. aeruginosa biofilm formation in association with 3-D lung epithelial cells in combination with the chelating agent EDTA. The dimeric derivative (lin-SB056-1)2-K demonstrated an enhanced biofilm-inhibitory activity as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death. Biofilm inhibition by (lin-SB056-1)2-K was reported both for the reference strain PAO1 and cystic fibrosis lung isolates of P. aeruginosa. In addition, using fluorescence microscopy, a significant decrease in biofilm-like structures associated with 3-D cells was observed after peptide exposure. Interestingly, effectiveness of (lin-SB056-1)2-K was also demonstrated in the wound model with a reduction of up to 1-Log unit in biofilm formation by P. aeruginosa PAO1 and wound isolates. Overall, combination treatment and peptide dendrimerization emerged as promising strategies to improve the efficacy of AMPs, especially under challenging host-mimicking conditions. Furthermore, the results of the present study underlined the importance of evaluating the biological properties of novel AMPs in in vivo-like model systems representative of specific infectious sites in order to make a more realistic prediction of their therapeutic success, and avoid the inclusion of unpromising peptides in animal studies and clinical trials.

Effects of amphipathic profile regularization on structural order and interaction with membrane models of two highly cationic branched peptides with ß-sheet propensity.

Antimicrobial peptides attracted increasing interest in last decades due to the rising concern of multi-drug resistant pathogens. Dendrimeric peptides are branched molecules with multiple copies of one peptide functional unit bound to the central core. Compared to linear analogues, they usually show improved activity and lower susceptibility to proteases. Knowledge of structure-function relationship is fundamental to tailor their properties. This work is focused on SB056, the smallest example of dendrimeric peptide, whose amino acid sequence is WKKIRVRLSA. Two copies are bound to the α- and ε- nitrogen of one lysine core. An 8-aminooctanamide was added at the C-terminus to improve membrane affinity. Its propensity for ß-type structures is also interesting, since helical peptides were already thoroughly studied. Moreover, SB056 maintains activity at physiological osmolarity, a typical limitation of natural peptides. An optimized analogue with improved performance was designed, ß-SB056, which differs only in the relative position of the first two residues (KWKIRVRLSA). This produced remarkable differences. Structure order and aggregation behavior were characterized by using complementary techniques and membrane models with different negative charge. Infrared spectroscopy showed different propensity for ordered ß-sheets. Lipid monolayers' surface pressure was measured to estimate the area/peptide and the ability to perturb lipid packing. Fluorescence spectroscopy was applied to compare peptide insertion into the lipid bilayer. Such small change in primary structure produced fundamental differences in their aggregation behavior. A regular amphipathic peptide's primary structure was responsible for ordered ß-sheets in a charge independent fashion, in contrast to unordered aggregates formed by the former analogue.

Correction: Energy expenditure in caving.

[This corrects the article DOI: 10.1371/journal.pone.0170853.].

Generation of Persister Cells of Pseudomonas aeruginosa and Staphylococcus aureus by Chemical Treatment and Evaluation of Their Susceptibility to Membrane-Targeting Agents.

Persister cells (PCs) are a subset of dormant, phenotypic variants of regular bacteria, highly tolerant to antibiotics. Generation of PCs in vivo may account for the recalcitrance of most chronic infections to antimicrobial treatment and demands for the identification of new antimicrobial agents able to target such cells. The present study explored the possibility to obtain in vitro PCs of Pseudomonas aeruginosa and Staphylococcus aureus at high efficiency through chemical treatment, and to test their susceptibility to structurally different antimicrobial peptides (AMPs) and two clinically used peptide-based antibiotics, colistin and daptomycin. The main mechanism of action of these molecules (i.e., membrane-perturbing activity) renders them potential candidates to act against dormant cells. Exposure of stationary-phase cultures to optimized concentrations of the uncoupling agent cyanide m-chlorophenylhydrazone (CCCP) was able to generate at high efficiency PCs exhibiting an antibiotic-tolerant phenotype toward different classes of antibiotics. The metabolic profile of CCCP-treated bacteria was investigated by monitoring bacterial heat production through isothermal microcalorimetry and by evaluating oxidoreductase activity by flow cytometry. CCCP-pretreated bacteria of both bacterial species underwent a substantial decrease in heat production and oxidoreductase activity, as compared to the untreated controls. After CCCP removal, induced persisters showed a delay in heat production that correlated with a lag phase before resumption of normal growth. The metabolic reactivation of bacteria coincided with their reversion to an antibiotic-sensitive phenotype. Interestingly, PCs generated by CCCP treatment resulted highly sensitive to three different membrane-targeting AMPs at levels comparable to those of CCCP-untreated bacteria. Colistin was also highly active against PCs of P. aeruginosa, while daptomycin killed PCs of S. aureus only at concentrations 32 to 64-fold higher than those of the tested AMPs. In conclusion, CCCP treatment was demonstrated to be a suitable method to generate in vitro PCs of medically important bacterial species at high efficiency. Importantly, unlike conventional antibiotics, structurally different AMPs were able to eradicate PCs suggesting that such molecules might represent valid templates for the development of new antimicrobials active against persisters.

The Semi-Synthetic Peptide Lin-SB056-1 in Combination with EDTA Exerts Strong Antimicrobial and Antibiofilm Activity against Pseudomonas aeruginosa in Conditions Mimicking Cystic Fibrosis Sputum.

Pseudomonas aeruginosa is a major cause of chronic lung infections in cystic fibrosis (CF) patients. The ability of the bacterium to form biofilms and the presence of a thick and stagnant mucus in the airways of CF patients largely contribute to antibiotic therapy failure and demand for new antimicrobial agents able to act in the CF environment. The present study investigated the anti-P. aeruginosa activity of lin-SB056-1, a recently described semi-synthetic antimicrobial peptide, used alone and in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA). Bactericidal assays were carried out in standard culture conditions and in an artificial sputum medium (ASM) closely resembling the CF environment. Peptide's structure and interaction with large unilamellar vesicles in media with different ionic strengths were also investigated through infrared spectroscopy. Lin-SB056-1 demonstrated fast and strong bactericidal activity against both mucoid and non-mucoid strains of P. aeruginosa in planktonic form and, in combination with EDTA, caused significant reduction of the biomass of P. aeruginosa mature biofilms. In ASM, the peptide/EDTA combination exerted a strong bactericidal effect and inhibited the formation of biofilm-like structures of P. aeruginosa. Overall, the results obtained highlight the potential of the lin-SB056-1/EDTA combination for the treatment of P. aeruginosa lung infections in CF patients.

Cytotoxic peptides with insulin-releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host-defense peptides differing by a single amino acid residue belonging to the brevinin-1 family (brevinin-1ITa and -1ITb), a peptide belonging to the temporin family (temporin-ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine-sulphoxide forms of brevinin-1ITa and -1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin-1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non-small cell lung adenocarcinoma A549 cells (LC50  = 18 µM), breast adenocarcinoma MDA-MB-231 cells (LC50  = 8 µM) and colorectal adenocarcinoma HT-29 cells (LC50  = 18 µM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC50  = 7 µM). Temporin-ITa (VFLGAIAQALTSLLGKL.NH2 ) was between three and fivefold less potent against these cells. Brevinin-1ITa inhibited growth of both Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin-ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, but brevinin-1ITa was cytotoxic to the cells at concentrations ≥3 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Antimicrobial Dendrimeric Peptides: Structure, Activity and New Therapeutic Applications.

Microbial resistance to conventional antibiotics is one of the most outstanding medical and scientific challenges of our times. Despite the recognised need for new anti-infective agents, however, very few new drugs have been brought to the market and to the clinic in the last three decades. This review highlights the properties of a new class of antibiotics, namely dendrimeric peptides. These intriguing novel compounds, generally made of multiple peptidic sequences linked to an inner branched core, display an array of antibacterial, antiviral and antifungal activities, usually coupled to low haemolytic activity. In addition, several peptides synthesized in oligobranched form proved to be promising tools for the selective treatment of cancer cells.

The urinary 1 H-NMR metabolomics profile of an italian autistic children population and their unaffected siblings.

Autism spectrum disorders (ASD) make a dishomogeneous group of psychiatric diseases having either genetic and environmental components, including changes of the microbiota. The rate of diagnosis, based on a series of psychological tests and observed behavior, dramatically increased in the past few decades. Currently, no biological markers are available and the pathogenesis is not defined. The purpose of this study was to evaluate the potential use of 1 H-NMR metabolomics to analyze the global biochemical signature of ASD patients (n = 21) and controls (n = 21), these being siblings of autistic patients. A multivariate model has been used to extrapolate the variables of importance. The discriminating urinary metabolites were identified; in particular, significantly increased levels of hippurate, glycine, creatine, tryptophan, and d-threitol and decreased concentrations of glutamate, creatinine, lactate, valine, betaine, and taurine were observed in ASD patients. Based on the identified discriminant metabolites, the attention was focused on two possible mechanisms that could be involved in ASD: oxidative stress conditions and gut microflora modifications. In conclusion, nuclear magnetic resonance-based metabolomics analysis of the urine seems to have the potential for the identification of a metabolic fingerprint of ASD phenotypes and appears to be suitable for further investigation of the disease mechanisms. Autism Res 2017. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. Autism Res 2017, 10: 1058-1066. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Energy expenditure in caving.

The aim of this study was to determine the energy expenditure of a group of cavers of both genders and different ages and experience during a 10 hour subterranean exploration, using portable metabolimeters. The impact of caving activity on body composition and hydration were also assessed through bioelectrical impedance, and nutritional habits of cavers surveyed. During cave activity, measured total energy expenditure (TEE) was in the range 225-287 kcal/h for women-men (MET = 4.1), respectively; subjects had an energy intake from food in the range 1000-1200 kcal, thus inadequate to restore lost calories. Bayesian statistical analysis estimated the effect of predictive variables on TEE, revealing that experienced subjects had a 5% lower TEE than the less skilled ones and that women required a comparatively larger energy expenditure than men to perform the same task. BIVA (bioelectrical impedance vector analysis) showed that subjects were within the range of normal hydration before and after cave activity, but bioelectrical changes indicated a reduction of extracellular water in men, which might result in hypo-osmolal dehydration in the case of prolonged underground exercise. All these facts should be considered when planning cave explorations, preparing training programs for subjects practising caving, and optimizing a diet for cavers. Further, information gathered through this study could be of value to reduce accidents in caves related to increase in fatigue.

Physical Capacity and Energy Expenditure of Cavers.

Caves are an extreme environment for humans because of the high humidity, mud, darkness, and slippery conditions. Explorations can last many hours or even days, and require extensive climbing and ropework. Very little is known about the physical capacity of cavers and their energy expenditure (EE) during caving. The physical capacity of 17 (7 females) expert cavers (age 43.9 ± 7.3 years) was assessed during an incremental cycle-ergometer test (IET) with gas exchange analysis. Moreover, a wearable metabolic band (Armband Fit Core) was used to estimate their EE during caving. In terms of physical capacity, the IET showed that cavers had a maximum oxygen uptake (VO2max) of 2,248.7 ± 657.8 ml·min-1 (i.e., 32.4 ± 6.4 ml·kg-1·min-1), while anaerobic threshold (AT) occurred on average at 74.5% of VO2max. Results from caving sessions provided an average time spent in cave of 9.4 ± 1.2 h while the average EE was 268.8 ± 54.8 kcal·h-1, which corresponded to about 40% of VO2max measured during IET. A mean distance of 10.6 ± 2.2 km was covered by subjects. Data from the present investigation provide evidence that cavers have a level of aerobic physical capacity only slightly higher than that of sedentary people, thereby suggesting that a high aerobic fitness is not needed by cavers. Moreover, during caving the EE was on average well below the level of AT. However, in absolute terms, the total EE was elevated (i.e., 2,672.3 ± 576 kcal in total) due to the long time spent in caving.

Peering into the Mediterranean black box: Lactifluus rugatus ectomycorrhizas on Cistus.

We describe the morpho-anatomical features of the ectomycorrhizas (ECMs) formed by Lactifluus rugatus on Cistus, a genus of about 20 species of woody shrubs typical of the Mediterranean maquis. The description of L. rugatus mycorrhizas on Cistus is the first ECM description of a species belonging to Lactifluus subgen. Pseudogymnocarpi. The ECM identity was verified through molecular tools. Anatomically, the characteristic of L. rugatus mycorrhiza is the presence of abundant, long "bottle-shaped" cystidia on mantle surface. Indeed, the overwhelming majority of milkcap mycorrhizas are acystidiate. This is the third Lactarius/Lactifluus mycorrhiza to have been described associated with Cistus, the others being Lactarius cistophilus and L. tesquorum. The phylogenetic distance between all these taxa is reflected by the diversity of the principal features of their ECMs, which share host-depending ECM features known for Cistus, but are otherwise distinguishable on the host roots. Comparison of Lactifluus rugatus ECM with those formed by L. vellereus and L. piperatus on Fagus reveals elevated intrageneric diversity of mycorrhizal structures. Such a diversity is supported by analysis of ITS sequences of relevant species within European Lactifluus species. Our study extends knowledge of Cistus mycorrhizal biology and confirms the informative value of mycorrhizal structures in understanding phylogenetic relationships in ECM fungi.