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The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these BGCs across the Fungal Kingdom. We amalgamated a pipeline of tools to predict BGCs based on shared promoter motifs and located 3800 ICS BGCs in 3300 genomes, making ICS BGCs the fifth largest class of specialized metabolites compared to canonical classes found by antiSMASH. ICS BGCs are not evenly distributed across fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the ICS dit1/2 gene cluster family (GCF), which was prior only studied in yeast, is present in ∼30% of all Ascomycetes. The dit variety ICS exhibits greater similarity to bacterial ICS than other fungal ICS, suggesting a potential convergence of the ICS backbone domain. The evolutionary origins of the dit GCF in Ascomycota are ancient and these genes are diversifying in some lineages. Our results create a roadmap for future research into ICS BGCs. We developed a website (https://isocyanides.fungi.wisc.edu/) that facilitates the exploration and downloading of all identified fungal ICS BGCs and GCFs., (Published by Oxford University Press on behalf of Nucleic Acids Research 2023.)

Ascomycota and basidiomycota fungi are prolific producers of biologically active natural products. Fungal natural products exhibit remarkable structural diversity and complexity, which are generated by the enzymes involved in their biosynthesis. After the formation of core skeletons, oxidative enzymes play a critical role in converting them into mature natural products. Besides simple oxidations, more complex transformations, such as multiple oxidations by single enzymes, oxidative cyclization, and skeletal rearrangement, are often observed. Those oxidative enzymes are of significant interest for the identification of new enzyme chemistry and have the potential to be biocatalysts for the synthesis of complex molecules. This review presents selected examples of unique oxidative transformations that have been found in the biosynthesis of fungal natural products. The development of strategies for refactoring the fungal biosynthetic pathways with an efficient genome-editing method is also introduced., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of moulds using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in a multicenter context. For that purpose, three Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5% and 94.8% and the score values were 1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (F. proliferatum, n = 1; T.interdigitale, n = 2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Endophytic fungi are broadly dispersed residing inside plant tissues and have been demonstrated as a treasure for bioactive natural products. Unexplored harsh and heavy metal contaminant habitat of Avicennia marina may have diverse and potential fungal association. Therefore, this work aimed to isolate the culturable fungal endophytes associated with leaves of A. marina and to evaluate their medical potentialities. Seventeen isolates of endophyte fungi were isolated from healthy leaves and their antimicrobial activities were evaluated. Results showed that isolates had activity against micro-organisms in addition to their antioxidant activity produced a variety of phenolic compounds, besides exhibited a lowest cytotoxicity against ATCC-CCL-81 cell line. Consequently, selected endophytic fungal isolates were identified genetically as Chaetomium sp., Chaetomium madrasense, Chaetomium sp., Chaetomium globosum, Aspergillus hiratsukae, Aspergillus ochraceus, Alternaria tenuissima and Curvularia lunata with gene bank accession numbers MT089951, MT089952, MT089953, MT089954, MT089955, MT089956, MT089957 and MT089958 respectively. The most potent fungus extract was analysed using Gas chromatography-mass spectrometry which verified the presence of numerous bioactive compounds. These findings confirmed that new endophytic fungal strains derived from A. marina thrive in harsh ecosystem produce bioactive metabolites which can be recommended as a novel source for drug discovery., (© 2020 The Society for Applied Microbiology.)

Stable, long-term interactions between fungi and algae or cyanobacteria, collectively known as lichens, have repeatedly evolved complex architectures with little resemblance to their component parts. Lacking any central scaffold, the shapes they assume are casts of secreted polymers that cement cells into place, determine the angle of phototropic exposure and regulate water relations. A growing body of evidence suggests that many lichen extracellular polymer matrices harbor unicellular, non-photosynthesizing organisms (UNPOs) not traditionally recognized as lichen symbionts. Understanding organismal input and uptake in this layer is key to interpreting the role UNPOs play in lichen biology. Here, we review both polysaccharide composition determined from whole, pulverized lichens and UNPOs reported from lichens to date. Most reported polysaccharides are thought to be structural cell wall components. The composition of the extracellular matrix is not definitively known. Several lines of evidence suggest some acidic polysaccharides have evaded detection in routine analysis of neutral sugars and may be involved in the extracellular matrix. UNPOs reported from lichens include diverse bacteria and yeasts for which secreted polysaccharides play important biological roles. We conclude by proposing testable hypotheses on the role that symbiont give-and-take in this layer could play in determining or modifying lichen symbiotic outcomes., (© FEMS 2020.)

CD154+ mould-reactive T cells were proposed as a novel biomarker in the diagnosis of invasive mycoses. As PBMC-based protocols for flow cytometric quantification of these cells are logistically challenging and susceptible to preanalytic delays, this study evaluated and optimized a whole blood-based method for the detection of mould-reactive T cells. Blood collection tubes containing costimulatory antibodies and Aspergillus fumigatus mycelial lysates were inoculated with heparinized whole blood from healthy adults, and detection rates of CD154+/CD4+A. fumigatus reactive T cells were compared with PBMC-based detection using samples from the same donors. In contrast to the PBMC-based method, double costimulation with αCD28 and αCD49d was crucial for reliable whole blood stimulation. Optimizing stimulation schemes for both matrixes, significantly higher specific T-cell detection rates were achieved by the whole blood-based method, whereas the unspecific background stimulation remained low. MHC II-dependent CD154+ upregulation was demonstrated for both matrixes. Excellent correlation and reproducible conversion factors between whole blood and PBMC-based results were observed. Using frozen ready-to-use test tubes containing costimulatory antibodies and lysates, detection rates of specific T cells were comparable to freshly prepared blood collection tubes. The optimized whole blood-based protocol was also used to detect Rhizopus arrhizus and Rhizomucor pusillus reactive T cells, resulting in 1.5- to 2.7-fold higher detection rates compared with PBMC-based measurement. In summary, the whole blood protocol is a robust, highly sensitive, and cost-effective method for mould-reactive T-cell quantification, allowing for point-of-care sample stimulation and contributing to better assay standardization in multi-centre evaluation of mould reactive T-cell quantification., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Spices are very important for cuisines around the world as well as for health enhancement. The Egyptians, Chinese, and Indians have used spices in medicinal remedies and procedures starting in around 2000 BC. Through the centuries, spices have found use as food ingredients to modify the aroma and taste of the final products; however, some spices are suitable substrates for mold growth and mycotoxin development, which could be detrimental to human and animal health. This report covers regulatory control of mycotoxins in food and spices by means of monitoring and regulatory limits, sampling and analysis, management, and prevention of mycotoxins from plant growth (preharvest) through harvest and postharvest as well as decontamination for mycotoxins when necessary. There is no one single-best strategy that can solve mycotoxin contamination problems, but a well-designed and integrated plan of all these strategies could result in a substantial reduction of mycotoxins in spices to regulation safety levels.

The plasma membrane (PM) performs a plethora of physiological processes, the coordination of which requires spatial and temporal organization into specialized domains of different sizes, stability, protein/lipid composition and overall architecture. Compartmentalization of the PM has been particularly well studied in the yeast Saccharomyces cerevisiae, where five non-overlapping domains have been described: The Membrane Compartments containing the arginine permease Can1 (MCC), the H+-ATPase Pma1 (MCP), the TORC2 kinase (MCT), the sterol transporters Ltc3/4 (MCL), and the cell wall stress mechanosensor Wsc1 (MCW). Additional cortical foci at the fungal PM are the sites where clathrin-dependent endocytosis occurs, the sites where the external pH sensing complex PAL/Rim localizes, and sterol-rich domains found in apically grown regions of fungal membranes. In this review, we summarize knowledge from several fungal species regarding the organization of the lateral PM segregation. We discuss the mechanisms of formation of these domains, and the mechanisms of partitioning of proteins there. Finally, we discuss the physiological roles of the best-known membrane compartments, including the regulation of membrane and cell wall homeostasis, apical growth of fungal cells and the newly emerging role of MCCs as starvation-protective membrane domains., (© FEMS 2019.)

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microorganisms in clinical laboratories because it is rapid, relatively simple to use, accurate, and can be used for a wide number of microorganisms. Several studies have demonstrated the utility of this technique in the identification of yeasts; however, its performance is usually improved by the extension of the database. Here we developed an in-house database of 143 strains belonging to 42 yeast species in the MALDI Biotyper platform, and we validated the extended database with 388 regional strains and 15 reference strains belonging to 55 yeast species. We also performed an intra- and interlaboratory study to assess reproducibility and analyzed the use of the cutoff values of 1.700 and 2.000 to correctly identify at species level. The creation of an in-house database that extended the manufacturer's database was successful in view of no incorrect identification was introduced. The best performance was observed by using the extended database and a cutoff value of 1.700 with a sensitivity of .94 and specificity of .96. A reproducibility study showed utility to detect deviations and could be used for external quality control. The extended database was able to differentiate closely related species and it has potential in distinguishing the molecular genotypes of Cryptococcus neoformans and Cryptococcus gattii.

In this study we evaluated the capacity of MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) to identify clinical mould isolates. We focused on two aspects of MALDI-TOF MS identification: the sample processing and the database. Direct smearing of the sample was compared with a simplified processing consisting of mechanical lysis of the moulds followed by a protein extraction step. Both methods were applied to all isolates and the Filamentous Fungi Library 1.0 (Bruker Daltonics) was used for their identification. This approach allowed the correct species-level identification of 25/34 Fusarium spp. and 10/10 Mucor circinelloides isolates using the simplified sample processing. In addition, 7/34 Fusarium spp. and 1/21 Pseudallescheria/Scedosporium spp. isolates were correctly identified at the genus level. The remaining isolates-60-could not be identified using the commercial database, mainly because of the low number of references for some species and the absence of others. Thus, an in-house library was built with 63 local isolates previously characterized using DNA sequence analysis. Its implementation allowed the accurate identification at the species level of 94 isolates (91.3%) and the remaining nine isolates (8.7%) were correctly identified at the genus level. No misidentifications at genus level were detected. In conclusion, with improvements of both the sample preparation and the feeding of the database, MALDI-TOF MS is a reliable, ready to use method to identify moulds of clinical origin in an accurate, rapid, and cost-effective manner.

Peptides with the inhibitor cystine knot (ICK) motif are extensively present in animals and plants where they exert a diversity of biological functions. However, few studies have been undertaken on this class of peptides in fungi. In this work, we identify a total of 386 fungal ICK peptides and proteins containing this motif by computational data mining of fungal genome databases, which exhibit 14 different exon-intron structures. According to their domain architectures, these proteins are classified into three distinct structural types, including single domains, tandem repeat domains and fusion domains, in which six families belonging to single or tandem repeat domains show remarkable sequence similarity to those from animals and plants, suggesting their orthologous relationship. Extremely high molecular diversity in fungal ICKs might be attributable to different genetic mechanisms, such as gene/domain duplication and fusion. This work not only enlarges the number of ICK peptides in multicellular organisms, but also uncovers their complex evolutionary history in a specific lineage.

This study aims to obtain secondary metabolites extracts from filamentous fungi isolated from soil and marine sediments from Antarctic ecosystems and to assess its potential antibacterial activity on Xanthomonas euvesicatoria and Xanthomonas axonopodis pv. passiflorae (phytopathogenic bacteria causing diseases in pepper and tomato and passionfruit, respectively). Among the 66 crude intracellular and extracellular extracts obtained from fungi recovered from soil and 79 obtained from marine sediment samples, 25 showed the ability to prevent the growth of X. euvesicatoria in vitro and 28 showed the ability to prevent the growth of X. axonopodis pv. passiflorae in vitro. Intracellular and extracellular extracts from soil fungi inhibited around 97% of X. euvesicatoria and 98% of X. axonopodis pv. passiflorae at 2·1 mg ml -1 . The average inhibition rates against X. euvesicatoria and X. axonopodis pv. passiflorae for intracellular and extracellular extracts from marine sediments fungi were around 96 and 97%, respectively, at 3·0 mg ml -1 . Extracts containing secondary metabolites with antimicrobial activity against X. euvesicatoria and X. axonopodis pv. passiflorae were obtained, containing possible substitutes for the products currently used to control these phytopathogens., Significance and Impact of the Study: Micro-organisms from extreme ecosystems, such as the Antarctic ecosystem, need to survive in harsh conditions with low temperatures, low nutrients and high UV radiation. Micro-organisms adapt to these conditions evolving diverse biochemical and physiological adaptations essential for survival. All this makes these micro-organisms a rich source of novel natural products based on unique chemical scaffolds. Discovering novel bioactive compounds is essential because of the rise in antibiotic-resistant micro-organisms and the emergence of new infections. Fungi from Antarctic environments have been proven to produce bioactive secondary metabolites against various micro-organisms, but few studies have shown activity against Xanthomonas phytopathogens., (© 2018 The Society for Applied Microbiology.)

Interest in endophytes as natural sources for new medicines was inspired by the discovery of paclitaxel-producing endophytic fungi. This study investigated the anti-cancer activity of extracts of endophytes isolated from two Australian plants, Eremophila longifolia (EL) and Eremophila maculata (EM). Endophytes were isolated from surface-sterilised leaf tissue, grown as pure cultures and identified by sequencing of Internal Transcribed Spacer (ITS) regions of the ribosomal DNA. To determine cytotoxicity, two leukaemic (MOLT-4, T-cell leukaemia; PreB-697, Pre-B leukaemia), a lung cancer cell line (A549) and a normal human fibroblast cell line were treated with endophyte extracts to assess cytotoxicity in relation to alternariol monomethyl ether (AME) and alternariol (AOH). Endophyte extracts that showed cell cytotoxicity were analysed by UV-HPLC to determine the metabolites. Pure AME and AOH, three extracts form Alternaria sp. (EM-6, EM-7 and EM-9) and one from Preussia minima (EL-14) were cytotoxic to the cancer cell lines. All cytotoxic endophytes contained AME and AOH, the most cytotoxic endophyte EM-6 also contained two unique peaks. These data indicate that these four endophyte extracts may have anti-cancer properties due to the presence of AME and AOH; however, the unique compounds found in the EM-6 extract may be exclusively cytotoxic and warrant further investigation.

The last years there has been a significant rise in the number of publications in the international literature that deal with the production of lipids by microbial sources (the 'single cell oils; SCOs' that are produced by the so-called 'oleaginous' micro-organisms). In the first part of the present review article, a general overview of the oleaginous micro-organisms (mostly yeasts, algae and fungi) and their potential upon the production of SCOs is presented. Thereafter, physiological and kinetic events related with the production of, mostly, yeast and fungal lipids when sugars and related substrates like polysaccharides, glycerol, etc. (the de novo lipid accumulation process) or hydrophobic substrates like oils and fats (the ex novo lipid accumulation process) were employed as microbial carbon sources, are presented and critically discussed. Considerations related with the degradation of storage lipid that had been previously accumulated inside the cells, are also presented. The interplay of the synthesis of yeast and fungal lipids with other intracellular (i.e. endopolysaccharides) or extracellular (i.e. citric acid) secondary metabolites synthesized is also presented. Finally, aspects related with the lipid extraction and lipidome analysis of the oleaginous micro-organisms are presented and critically discussed., (© 2017 The Society for Applied Microbiology.)

Metabolic capabilities of microorganisms include the production of secondary metabolites (e.g. antibiotics). The analysis of microbial volatile organic compounds (mVOCs) is an emerging research field with huge impact on medical, agricultural and biotechnical applied and basic science. The mVOC database (v1) has grown with microbiome research and integrated species information with data on emitted volatiles. Here, we present the mVOC 2.0 database with about 2000 compounds from almost 1000 species and new features to work with the database. The extended collection of compounds was augmented with data regarding mVOC-mediated effects on plants, fungi, bacteria and (in-)vertebrates. The mVOC database 2.0 now features a mass spectrum finder, which allows a quick mass spectrum comparison for compound identification and the generation of species-specific VOC signatures. Automatic updates, useful links and search for mVOC literature are also included. The mVOC database aggregates and refines available information regarding microbial volatiles, with the ultimate aim to provide a comprehensive and informative platform for scientists working in this research field. To address this need, we maintain a publicly available mVOC database at: http://bioinformatics.charite.de/mvoc., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Metals are essential for life, and they play a central role in the struggle between infecting microbes and their hosts. In fact, an important aspect of microbial pathogenesis is the 'nutritional immunity', in which metals are actively restricted (or, in an extended definition of the term, locally enriched) by the host to hinder microbial growth and virulence. Consequently, fungi have evolved often complex regulatory networks, uptake and detoxification systems for essential metals such as iron, zinc, copper, nickel and manganese. These systems often differ fundamentally from their bacterial counterparts, but even within the fungal pathogens we can find common and unique solutions to maintain metal homeostasis. Thus, we here compare the common and species-specific mechanisms used for different metals among different fungal species-focusing on important human pathogens such as Candida albicans, Aspergillus fumigatus or Cryptococcus neoformans, but also looking at model fungi such as Saccharomyces cerevisiae or A. nidulans as well-studied examples for the underlying principles. These direct comparisons of our current knowledge reveal that we have a good understanding how model fungal pathogens take up iron or zinc, but that much is still to learn about other metals and specific adaptations of individual species-not the least to exploit this knowledge for new antifungal strategies., (© FEMS 2017.)

This study provided a reliable way to identify and estimate the ergosterol in farm environments, since it is renowned that it is a specific indicator for the occurrence of molds and yeasts. The quick valuation of exposure to airborne microorganisms is essential to assess the risk to which the health of employees is subjected in working places characterized by great humidity. From this view, it is worth estimating the total biomass of molds and yeast, including viable and non-viable forms, which may cause respiratory concerns to human. Air samples were collected with a passive method and the microbial growth was evaluated with a traditional counting method. At the same time, the ergosterol was quantified from yeasts and molds. In this way, the aerosol concentrations of molds and yeasts were compared by using the two methods, the cultivation on plates, as well as through ergosterol measurement by means of UV spectroscopy. Results showed, for the first time, a positive correlation between the amount of ergosterol and the yeast cells. Based on these outcomes, the ergosterol is a statistically significant biomarker to be used to control the air quality of indoor and outdoor farm spaces, by means of a simple and direct UV procedure., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Partially mycoheterotrophic plants are enriched in 13 C and 15 N compared to autotrophic plants. Here, it is hypothesized that the type of mycorrhizal fungi found in orchid roots is responsible for variation in 15 N enrichment of leaf tissue in partially mycoheterotrophic orchids., Methods: The genus Epipactis was used as a case study and carbon and nitrogen isotope abundances of eight Epipactis species, fungal sporocarps of four Tuber species and autotrophic references were measured. Mycorrhizal fungi were identified using molecular methods. Stable isotope data of six additional Epipactis taxa and ectomycorrhizal and saprotrophic basidiomycetes were compiled from the literature., Key Results: The 15 N enrichment of Epipactis species varied between 3·2 ± 0·8 ‰ ( E. gigantea ; rhizoctonia-associated) and 24·6 ± 1·6 ‰ ( E. neglecta ; associated with ectomycorrhizal ascomycetes). Sporocarps of ectomycorrhizal ascomycetes (10·7 ± 2·2 ‰) were significantly more enriched in 15 N than ectomycorrhizal (5·2 ± 4·0 ‰) and saprotrophic basidiomycetes (3·3 ± 2·1 ‰)., Conclusions: As hypothesized, it is suggested that the observed gradient in 15 N enrichment of Epipactis species is strongly driven by 15 N abundance of their mycorrhizal fungi; i.e. ɛ 15 N in Epipactis spp. associated with rhizoctonias < ɛ 15 N in Epipactis spp. with ectomycorrhizal basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes and basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes., (© The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Filamentous fungi are an unexplored source for the production of biosurfactants, but over a decade one of the most surface active molecules called hydrophobins was discovered. There are few techniques to determine the surface activity of fungi without any kind of manipulation that can affect the final results. In this work, we identified 33 strains of filamentous fungi isolated from banana rachis which may have potential in producing biosurfactants. Further, the production of surface active compounds by the strains was measured by two techniques. First, the surface tension of supernatants was evaluated in liquid cultures of the strains. We found that three strains belonging to the genus Fusarium, Penicillium and Trichoderma showed activity in the reduction of surface tension, which indicate a putative production of biosurfactants. Second, we measured the contact angle between the drop of water and the solid culture of strains to determine the surface activity of cells, classifying the strains as hydrophilic or hydrophobic. These techniques can be used as a quantitative measurement of the surface activity of fungi without cell manipulation., Significance and Impact of the Study: Biosurfactants are an alternative to petrochemical derivatives, and filamentous fungi are a promising source of these molecules. This work identified 33 strains of filamentous fungi in agroindustrial wastes. This is important because these results open the opportunity of finding new biosurfactants (hydrophobins) with unique properties. We propose the evaluation of surface tension in the supernatant as a quantitative screening to determine the production of biosurfactants from the strains of fungi., (© 2017 The Society for Applied Microbiology.)

Soils are complex ecosystems in which fungi and bacteria co-exist and interact. Fungal highways are a kind of interaction by which bacteria use fungal hyphae to disperse in soils. Despite the fact that fungal highways have been studied in laboratory models, the diversity of fungi and bacteria interacting in this way in soils is still unknown. Fungal highway columns containing two different culture media were used as a selective method to study the identity of fungi and bacteria able to migrate along the hyphae in three forest soils. Regardless of the soil type, fungi of the genus Mortierella (phylum Zygomycota) were selected inside the columns. In contrast, a diverse community of bacteria dominated by Firmicutes and Proteobacteria was observed. The results confirm the importance of bacteria affiliated to Burkholderia as potentially associated migrating bacteria in soils and indicate that other groups such as Bacillus and Clostridium are also highly enriched in the co-colonization of a new habitat (columns) associated to Mortierella. The diversity of potentially associated migrating bacteria brings a novel perspective on the indirect metabolic capabilities that could be favored by r-strategist fungi and supports the fact that these fungi should be considered as crucial actors in soil functioning., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Increase in invasive fungal infections over the past few years especially in immunocompromised patients prompted the search for new antifungal agents with improved efficacy. Current antifungal armoury includes very few effective drugs like Amphotericin B; new generation azoles, including voriconazole and posaconazole; echinocandins like caspofungin and micafungin to name a few. Azole class of antifungals which target the fungal cell membrane are the first choice of treatment for many years because of their effectiveness. As the fungal cell membrane is predominantly made up of sterols, glycerophospholipids and sphingolipids, the role of lipids in pathogenesis and target identification for improved therapeutics were largely pursued by researchers during the last few years. Present review focuses on cell membrane as an antifungal target with emphasis on membrane biogenesis, structure and function of cell membrane, cell membrane inhibitors, screening assays, recent advances and future prospects., (© 2016 The Society for Applied Microbiology.)

Moulds and arthroconidial yeasts are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens hastens initiation of targeted antifungal therapy, thereby improving the patients' prognosis. We describe a new strategy that enabled the identification of moulds and arthroconidial yeasts directly from positive blood cultures by MALDI-TOF mass spectrometry (MS). Positive blood cultures (BCs) with Gram staining showing hyphae and/or arthroconidia were prospectively selected and submitted to an in-house protein extraction protocol. Mass spectra were obtained by Vitek MS™ system, and identifications were carried out with in the research use only (RUO) mode with an extended database (SARAMIS™ [v.4.12] plus in-house database). Fusarium solani, Fusarium verticillioides, Exophiala dermatitidis, Saprochaete clavata, and Trichosporon asahii had correct species ID by MALDI-TOF MS analysis of positive BCs. All cases were related to critically ill patients with high mortality fungemia and direct ID from positive BCs was helpful for rapid administration of targeted antifungal therapy., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus.

In this study, we isolated 15 endophytic fungi from five Sudanese medicinal plants. Each fungal endophytic strain was identified by sequencing of internal transcribed spacer (ITS) regions of rDNA. Ethyl acetate extracts were prepared from each endophyte cultivated in vitro and tested for their respective antibacterial activities and antiproliferative activities against human cancer cells. Antibacterial screening was carried out against two bacterial strains: Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus, by the broth dilution method. Cell viability was evaluated by the MTT procedure after exposure of MCF7 breast cancer cells and HT29 or HCT116 human colon adenocarcinoma cells to each endophytic extract. Of interest, Byssochlamys spectabilis isolated from Euphorbia prostata showed cytotoxicity (IC50 = 1.51 ± 0.2 μg mL(-1)) against MCF7 cells, but had a low effect against HT29 or HCT116 cells (IC50 > 20 μg mL(-1)). Cladosporium cladosporioides 2, isolated from Vernonia amygdalina leaves, showed antiproliferative activities against MCF7 cells (IC50 = 10.5 ± 1.5 μg mL(-1)) only. On the other hand, B. spectabilis and Alternaria sp. extract had antibacterial activities against the S. aureus strain. The findings of this work revealed that endophytic fungi associated with medicinal plants from Sudan could be considered as an attractive source of new therapeutic compounds., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aim: To initiate a genetic and bioactivity-based screening programme of culturable endophytes to identify micro-organisms capable of producing bioactive polyketides and peptides., Methods and Results: Fungal endophytes were isolated from flowers, leaves and roots of Rhoeo spathacea, revealing a community consisting of Colletotrichum sp., Fusarium sp., Guignardia sp., Phomopsis sp., Phoma sp. and Microdochium sp. Genetic screening showed that all isolates had polyketide synthase (PKS) genes and most had nonribosomal peptide synthetase (NRPS) genes. Ethyl acetate extracts of the fungal isolates exhibited antiproliferative activity against at least one of the seven bacterial and mycobacterial test strains. Nuclear Magnetic Resonance -guided fractionation of the crude extract from a Fusarium sp. strain which exhibited strong antiproliferative activity against Mycobacterium tuberculosis resulted in the isolation of the polyketide javanicin. This compound was active against Myco. tuberculosis (MIC = 25 μg ml(-1)) and Mycobacterium phlei (MIC = 50 μg ml(-1))., Conclusions: The medicinal plant R. spathacea hosts a variety of fungal endophytes capable of producing antibacterial and antimycobacterial compounds. There is a positive correlation between the presence of PKS and/or NRPS encoding genes in endophytes and the bioactivity of their respective organic extracts., Significance and Impact of the Study: This is the first report on the fungal endophytic diversity of R. spathacea, and the isolation of an antimycobacterial compound from the plant which has been traditionally used for the treatment of tuberculosis symptoms., (© 2016 The Society for Applied Microbiology.)

The Carbohydrate Structure Databases (CSDBs, http://csdb.glycoscience.ru) store structural, bibliographic, taxonomic, NMR spectroscopic, and other data on natural carbohydrates and their derivatives published in the scientific literature. The CSDB project was launched in 2005 for bacterial saccharides (as BCSDB). Currently, it includes two parts, the Bacterial CSDB and the Plant&Fungal CSDB. In March 2015, these databases were merged to the single CSDB. The combined CSDB includes information on bacterial and archaeal glycans and derivatives (the coverage is close to complete), as well as on plant and fungal glycans and glycoconjugates (almost all structures published up to 1998). CSDB is regularly updated via manual expert annotation of original publications. Both newly annotated data and data imported from other databases are manually curated. The CSDB data are exportable in a number of modern formats, such as GlycoRDF. CSDB provides additional services for simulation of (1)H, (13)C and 2D NMR spectra of saccharides, NMR-based structure prediction, glycan-based taxon clustering and other., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Western flower thrips, Frankliniella occidentalis (Pergande), is one of the most destructive insect pests of greenhouse production systems with the ability to develop resistance to a wide variety of insecticides. A common resistance management strategy is rotating insecticides with different modes of action. By incorporating entomopathogenic organisms (fungi and bacteria), which have discrete modes of action compared to standard insecticides, greenhouse producers may preserve the effectiveness of insecticides used for suppression of western flower thrips populations. The objective of this study was to determine how different rotation programs that include entomopathogenic organisms (Beauveria bassiana, Isaria fumosoroseus, Metarhizium anisopliae, and Chromobacterium subtsugae) and commonly used standard insecticides (spinosad, chlorfenapyr, abamectin, and pyridalyl) may impact the population dynamics of western flower thrips adult populations by means of suppression. Eight-week rotation programs were applied to chrysanthemum, Dendranthema x morifolium plants and weekly counts of western flower thrips adults captured on yellow sticky cards were recorded as a means to evaluate the impact of the rotation programs. A final quality assessment of damage caused by western flower thrips feeding on foliage and flowers was also recorded. Furthermore, a cost comparison of each rotation program was conducted. Overall, insecticide rotation programs that incorporated entomopathogenic organisms were not significantly different than the standard insecticide rotation programs without entomopathogenic organisms in suppressing western flower thrips adult populations. However, there were no significant differences among any of the rotation programs compared to the water control. Moreover, there was no differential effect of the rotation programs on foliage and flower quality. Cost savings of up to 34% (in US dollars) are possible when including entomopathogenic organisms in the rotation program. Therefore, by incorporating entomopathogenic organisms into insecticide rotation programs, greenhouse producers can decrease costs without affecting suppression, as well as diminish selection pressure on western flower thrips adult populations, which may avoid or delay resistance development., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Among sulphated glycans, little is known about 3'-sulphation because of the lack of useful probes. In the course of molecular engineering of a fungal galectin from Agrocybe cylindracea, we found that a single substitution of Glu86 with alanine resulted in acquisition of specific binding for the 3'-sulpho-Galβ1-4GlcNAc structure. Extensive glyco-technological analysis revealed that this property was obtained in a 'loss-of-function' manner. Though this mutant (E86A) had low total affinity, it showed substantial binding to a naturally occurring N-glycan, of which the terminal galactose is 3-sulphated. Moreover, E86A specifically bound to HeLa cells, in which galactose-3-O-sulfotransferases (Gal3ST2 or Gal3ST3) were over-expressed., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Objectives: This study was conducted to evaluate the vasorelaxant effect of the fungal alkaloids malbrancheamides on pre-contracted rat aorta rings. Also, we explored the probable mode of action using experimental and theoretical docking studies., Methods: The vasorelaxant effect was assessed on rat aorta rings pre-contracted with noradrenaline (0.1 μm). The mechanism of action was evaluated using different inhibitors of the pathways involved in the vasorelaxation process, such as l-NAME, indomethacin, tetraethylammonium and atropine. The docking analyses were carried out with AutoDock 4.2 software using the crystallized structure of the cyclooxygenase domain of eNOS., Key Findings: Malbrancheamides (1-3) induced a significant vasorelaxant activity in a concentration- and endothelium-intact model in rat aorta rings, and a lesser effect in an endothelium-denuded model. Malbrancheamide-induced vasorelaxation was significantly weakened by pretreatment of endothelium-intact aortic rings with L-NAME (10 μm), indicating a nitrergic relaxant mechanism. Docking analysis predicted that 1-3 could activate eNOS throughout an allosteric fashion at C1 and C2 pockets., Conclusions: Experimental evidence revealed that malbrancheamides induced both endothelium-independent and endothelium-dependent relaxant effects. According to theoretical studies, it is feasible that the endothelium-independent relaxation exerted by malbrancheamide could be mediated by its calmodulin inhibitory properties throughout an interference with myosin light chain phosphorylation and a positive modulation of eNOS., (© 2015 Royal Pharmaceutical Society.)

Background: First introduced into clinical microbiology laboratories in Europe, MALDI-TOF MS is being rapidly embraced by laboratories around the globe. Although it has multiple applications, its widespread adoption in clinical microbiology relates to its use as an inexpensive, easy, fast, and accurate method for identification of grown bacteria and fungi based on automated analysis of the mass distribution of bacterial proteins., Content: This review provides a historical perspective on this new technology. Modern applications in the clinical microbiology laboratory are reviewed with a focus on the most recent publications in the field. Identification of aerobic and anaerobic bacteria, mycobacteria, and fungi are discussed, as are applications for testing urine and positive blood culture bottles. The strengths and limitations of MALDI-TOF MS applications in clinical microbiology are also addressed., Summary: MALDI-TOF MS is a tool for rapid, accurate, and cost-effective identification of cultured bacteria and fungi in clinical microbiology. The technology is automated, high throughput, and applicable to a broad range of common as well as esoteric bacteria and fungi. MALDI-TOF MS is an incontrovertibly beneficial technology for the clinical microbiology laboratory., (© 2014 American Association for Clinical Chemistry.)

The identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) relies mainly on a robust and extensive database of reference spectra. To this end, a large in-house library containing 760 strains and representing 472 species was built and evaluated on 390 clinical isolates by comparing MALDI-TOF MS with the classical identification method based on morphological observations. The use of MALDI-TOF MS resulted in the correct identification of 95.4% of the isolates at species level, without considering LogScore values. Taking into account the Brukers' cutoff value for reliability (LogScore >1.70), 85.6% of the isolates were correctly identified. For a number of isolates, microscopic identification was limited to the genus, resulting in only 61.5% of the isolates correctly identified at species level while the correctness reached 94.6% at genus level. Using this extended in-house database, MALDI-TOF MS thus appears superior to morphology in order to obtain a robust and accurate identification of filamentous fungi. A continuous extension of the library is however necessary to further improve its reliability. Indeed, 15 isolates were still not represented while an additional three isolates were not recognized, probably because of a lack of intraspecific variability of the corresponding species in the database., (© The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytochrome P450 (CYP) monooxygenase superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs has mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the fungal AP sequence space and to obtain an in-depth understanding of fungal AP evolution. Using a comprehensive phylogeny of approximately 700 AP sequences from the complete proteomes of 87 fungi and 20 nonfungal eukaryotes, 11 major clades of APs were defined of which clade I largely corresponds to the A1A subfamily of pepsin-archetype APs. Clade II largely corresponds to the A1B subfamily of nepenthesin-archetype APs. Remarkably, the nine other clades contain only fungal APs, thus indicating that fungal APs have undergone a large sequence diversification. The topology of the tree indicates that fungal APs have been subject to both "birth and death" evolution and "functional redundancy and diversification." This is substantiated by coclustering of certain functional sequence characteristics. A meta-analysis toward the identification of Cluster Determining Positions (CDPs) was performed in order to investigate the structural and biochemical basis for diversification. Seven CDPs contribute to the secondary structure of the enzyme. Three other CDPs are found in the vicinity of the substrate binding cleft. Tree topology, the large sequence variation among fungal APs, and the apparent functional diversification suggest that an amendment to update the current A1 AP classification based on a comprehensive phylogenetic clustering might contribute to refinement of the classification in the MEROPS peptidase database., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Marine-derived microbial secondary metabolites are promising potential sources of nontoxic antifouling agents. The search for environmentally friendly and low-toxic antifouling components guided us to investigate the antifouling potentials of eight novel fungal isolates from deep-sea sediments of the South China Sea. Sixteen crude ethyl acetate extracts of the eight fungal isolates showed distinct antibacterial activity against three marine bacteria (Loktanella hongkongensis UST950701-009, Micrococcus luteus UST950701-006 and Pseudoalteromonas piscida UST010620-005), or significant antilarval activity against larval settlement of bryozoan Bugula neritina. Furthermore, the extract of Aspergillus westerdijkiae DFFSCS013 displayed strong antifouling activity in a field trial lasting 4 months. By further bioassay-guided isolation, five antifouling alkaloids including brevianamide F, circumdatin F and L, notoamide C, and 5-chlorosclerotiamide were isolated from the extract of A. westerdijkiae DFFSCS013. This is the first report about the antifouling potentials of metabolites of the deep-sea-derived fungi from the South China Sea, and the first stage towards the development of non- or low-toxic antifouling agents from deep-sea-derived fungi.

Scents are well known to be emitted from flowers and animals. In nature, these volatiles are responsible for inter- and intra-organismic communication, e.g. attraction and defence. Consequently, they influence and improve the establishment of organisms and populations in ecological niches by acting as single compounds or in mixtures. Despite the known wealth of volatile organic compounds (VOCs) from species of the plant and animal kingdom, in the past, less attention has been focused on volatiles of microorganisms. Although fast and affordable sequencing methods facilitate the detection of microbial diseases, however, the analysis of signature or fingerprint volatiles will be faster and easier. Microbial VOCs (mVOCs) are presently used as marker to detect human diseases, food spoilage or moulds in houses. Furthermore, mVOCs exhibited antagonistic potential against pathogens in vitro, but their biological roles in the ecosystems remain to be investigated. Information on volatile emission from bacteria and fungi is presently scattered in the literature, and no public and up-to-date collection on mVOCs is available. To address this need, we have developed mVOC, a database available online at http://bioinformatics.charite.de/mvoc.

Despite widespread application of nucleic acid diagnostics, cultures remain integral in modern laboratories. Because cultures detect a large number of organism types, it is unlikely that they will disappear from clinical practice in the near future. Their downside is slow turn-around time, impacted by time to growth and identification of that growth. The latter is expedited using a new proteomic technology, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Vaults are very large oligomeric ribonucleoproteins conserved among a variety of species. The rat vault 3D structure shows an ovoid oligomeric particle, consisting of 78 major vault protein monomers, each of approximately 861 amino acids. Vaults are probably the largest ribonucleoprotein structures in eukaryote cells, being approximately 70 nm in length with a diameter of 40 nm--the size of three ribosomes and with a lumen capacity of 50 million Å(3). We use both protein sequences and inferred ancestral sequences for in silico virtual resurrection of tertiary and quaternary structures to search for vaults in a wide variety of eukaryotes. We find that the vault's phylogenetic distribution is widespread in eukaryotes, but is apparently absent in some notable model organisms. Our conclusion from the distribution of vaults is that they were present in the last eukaryote common ancestor but they have apparently been lost from a number of groups including fungi, insects, and probably plants. Our approach of inferring ancestral 3D and quaternary structures is expected to be useful generally.

Microorganisms have to cope with restricted iron bioavailability in most environmental habitats as well as during host colonization. The continuous struggle for iron has brought forth a plethora of acquisition and assimilation strategies that share several functional and mechanistic principles. One common theme is the utilization of high-affinity chelators for extracellular iron mobilization, generally known as siderophore-dependent iron acquisition. This basic strategy is related with another central aspect of microbial iron acquisition, which is the release of the mobilized iron from extracellular sources to allow its transfer and incorporation into metabolically active proteins. A variety of mechanisms which are often coupled with high-affinity uptake have evolved to facilitate the removal of iron from siderophore ligands; however, they differ in many key aspects including substrate specificities and release efficiencies. The most sophisticated iron release pathways comprise processes of specific hydrolysis and reduction of ferric siderophores, especially in the case of high-affinity iron complexes with greatly negative redox potentials that often represent crucial factors for virulence development in bacterial and fungal pathogens. During the following steps of iron utilization, the acquired metal is transferred through intracellular trafficking pathways which may include diverse storage compartments in order to be directed to cofactor assembly systems and to final protein targeting. Several of these iron channeling routes have been described recently and provide first insights into the later steps of iron assimilation that characterize an essential part of the cellular iron homeostasis network.

Until recently, microbial identification in clinical diagnostic laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The development of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) devices has revolutionized the routine identification of microorganisms in clinical microbiology laboratories by introducing an easy, rapid, high throughput, low-cost, and efficient identification technique. This technology has been adapted to the constraint of clinical diagnostic laboratories and has the potential to replace and/or complement conventional identification techniques for both bacterial and fungal strains. Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method. Similarly, the routine identification by MALDI-TOF MS of yeast isolates is reliable and much quicker than conventional techniques. Recent studies have shown that MALDI-TOF MS has also the potential to accurately identify filamentous fungi and dermatophytes, providing that specific standardized procedures are established for these microorganisms. Moreover, MALDI-TOF MS has been used successfully for microbial typing and identification at the subspecies level, demonstrating that this technology is a potential efficient tool for epidemiological studies and for taxonomical classification., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of β-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in-depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets.

Sarcoidosis is an inflammatory disease. Epidemiological and treatment studies suggest that fungi play a part in the pathogenesis. The aim of this work was to study the effect of fungal cell wall agents (FCWA) on the in vitro secretion of cytokines from peripheral blood monocytes from subjects with sarcoidosis and relate the results to fungal exposure at home and clinical findings. Subjects with sarcoidosis (n=22) and controls (n=20) participated. Peripheral blood mononuclear cells were stimulated with soluble or particulate β-glucan (S-glucan, P-glucan), chitin or lipopolysaccharide (LPS), whereafter tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 were measured. The severity of sarcoidosis was determined using a chest X-ray-based score. Serum cytokines (IL-2R, IL-6, IL-10 and IL-12) were determined. To measure domestic fungal exposure, air in the bedrooms was sampled on filters. N-acetylhexosaminidase (NAHA) on the filters was measured as a marker of fungal cell biomass. The induced secretion of cytokines was higher from peripheral blood mononuclear cells (PBMC) from subjects with sarcoidosis. P-glucan was more potent than S-glucan inducing a secretion. Chitin had a small effect. Among subjects with sarcoidosis there was a significant relation between the spontaneous PBMC production of IL-6, IL-10 and IL-12 and the NAHA levels at home. The P-glucan induced secretion of IL-12 was related to the duration of symptoms at the time of diagnosis. Their X-ray scores were related to an increased secretion of cytokines after stimulation with LPS or P-glucan. Subjects with sarcoidosis have a higher reactivity to FCWA in vitro and to home exposure. The influence of FCWA on inflammatory cells and their interference with the inflammatory defense mechanisms in terms of cytokine secretion could be important factors for the development of sarcoidosis., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Accurate species diagnosis in cases of fungal pneumonia may be hampered by environmental contamination and colonization resulting in false-positive results. Our novel approach for fungal species diagnostics combines fluorescent staining of mounted cryosections with the optical brightener Blankophor, laser capture microdissection and PCR amplification with subsequent sequencing of the first internal transcribed spacer region (ITS-1). Using clinical specimens from infected birds, we show that the procedure is suitable for species identification from single hyphae of intralesional filamentous fungi. Our data also suggest that multiple Aspergillus fumigatus strain infections may occur frequently in pulmonary aspergillosis of birds.

We describe an efficient SCHEMA recombination-based approach for screening homologous enzymes to identify stabilizing amino acid sequence blocks. This approach has been used to generate active, thermostable cellobiohydrolase class I (CBH I) enzymes from the 390 625 possible chimeras that can be made by swapping eight blocks from five fungal homologs. Constructing and characterizing the parent enzymes and just 32 'monomeras' containing a single block from a homologous enzyme allowed stability contributions to be assigned to 36 of the 40 blocks from which the CBH I chimeras can be assembled. Sixteen of 16 predicted thermostable chimeras, with an average of 37 mutations relative to the closest parent, are more thermostable than the most stable parent CBH I, from the thermophilic fungus Talaromyces emersonii. Whereas none of the parent CBH Is were active >65°C, stable CBH I chimeras hydrolyzed solid cellulose at 70°C. In addition to providing a collection of diverse, thermostable CBH Is that can complement previously described stable CBH II chimeras (Heinzelman et al., Proc. Natl Acad. Sci. USA 2009;106:5610-5615) in formulating application-specific cellulase mixtures, the results show the utility of SCHEMA recombination for screening large swaths of natural enzyme sequence space for desirable amino acid blocks.

Many volatile organic compounds (VOCs) are found in indoor environment as products of microbial metabolism. In damp indoor environments, fungi are associated with poor air quality. Some epidemiological studies have suggested that microbial VOCs have a negative impact on human health. Our study was designed to provide a reductionist approach toward studying fungal VOC-mediated toxicity using the inexpensive model organism, Drosophila melanogaster, and pure chemical standards of several important fungal VOCs. Low concentrations of the following known fungal VOCs, 0.1% of 1-octen-3-ol and 0.5% of 2-octanone; 2,5 dimethylfuran; 3-octanol; and trans-2-octenal, caused locomotory defects and changes in green fluorescent protein (GFP)- and antigen-labeled dopaminergic neurons in adult D. melanogaster. Locomotory defects could be partially rescued with L-DOPA. Ingestion of the antioxidant, vitamin E, improved the survival span and delayed the VOC-mediated changes in dopaminergic neurons, indicating that the VOC-mediated toxicity was due, in part, to generation of reactive oxygen species.

Aims: A sterile red fungus (SRF) isolated from cortices of roots of both wheat (Triticum aestivum cv. Gamenya) and ryegrass (Lolium rigidum cv. Wimmera) was found to protect the hosts from phytopathogens and promote plant growth. In this work, the major secondary metabolites produced by this SRF were analysed, and their antibiotic and plant-growth-promoting activities investigated., Methods and Results: Two main compounds, veratryl alcohol (VA) and 4-(hydroxymethyl)-quinoline, were isolated from the culture filtrate of the fungus. In antifungal assays, VA inhibited the growth of Sclerotinia sclerotiorum and Pythium irregulare even at low amounts, while high concentrations (>100 microg per plug) of 4-(hydroxymethyl)-quinoline were needed. Both metabolites revealed weak inhibition of Rhizoctonia solani. Furthermore, both compounds showed a growth promotion activity on canola (Brassica napus) seedlings used as bioassays., Conclusions: Isolation and characterization of the main secondary metabolites from culture filtrates of a root-inhabiting sterile fungus are reported. The biological assays indicate that these secondary metabolites may have a role in both plant growth regulation and antifungal activity., Significance and Impact of the Study: This study provides a better understanding of the metabolism of a cortical fungus that may have a useful role in the biological suppression of root-infecting soil-borne plant pathogens.

Aim: To determine whether assessing the penetration of solutions with different concentrations of ethanol (alcohol percentage test: APT) on fungal surfaces is effective in characterization of hydrophobicity on fungal surfaces., Methods and Results: APT and contact angle (CA) measurements were conducted on nine hydrophobic and two hydrophilic fungal strains from the phyla of Ascomycota, Basidiomycota and Zygomycota. There was a strong positive correlation (R(2) = 0.95) between the APT and CA measurements from eight of the nine hydrophobic stains (four pathogenic and mycotoxigenic Fusarium taxa, one melanosporaceous biotrophic taxon, Alternaria sp, Penicillium aurantiogriseum and Cladosporium cladosporioides). Hydrophilic control strains, Mortierella hyalina and Laccaria laccata, had CAs <90 degrees and no measurable degree of hydrophobicity using the APT method., Conclusions: The APT method was effective in measuring the degree of hydrophobicity and can be conducted on different zones of fungal growth., Significance and Impact of the Study: Characterization of fungal surface hydrophobicity is important for understanding of its particular role and function in fungal morphogenesis and pathogenesis. APT is a simple method that can be utilized for fungal hydrophobicity measurements when CA cannot be measured because of obscured view from aerial mycelia growth.

Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF ICMS) is coming of age for the identification and characterization of fungi. The procedure has been used extensively with bacteria. UV-absorbing matrices function as energy mediators that transfer the absorbed photoenergy from an irradiation source to the surrounding sample molecules, resulting in minimum fragmentation. A surprisingly high number of fungal groups have been studied: (i) the terverticillate penicillia, (ii) aflatoxigenic, black and other aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting fungi (e.g. Serpula lacrymans) and (v) dermatophytes. The technique has been suggested for optimizing quality control of fungal Chinese medicines (e.g. Cordyceps). MALDI-TOF ICMS offers advantages over PCR. The method is now used in taxonomic assessments (e.g. Trichoderma) as distinct from only strain characterization. Low and high molecular mass natural products (e.g. peptaibols) can be analysed. The procedure is rapid and requires minimal pretreatment. However, issues of reproducibility need to be addressed further in terms of strains of species tested and between run variability. More studies into the capabilities of MALDI-TOF ICMS to identify fungi are required.