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Background Numerous functional genomics approaches have been developed to study the model organism yeast, Saccharomyces cerevisiae, with the aim of systematically understanding the biology of the cell. Some of these techniques are based on yeast growth differences under different conditions, such as those generated by gene mutations, chemicals or both. Manual inspection of the yeast colonies that are grown under different conditions is often used as a method to detect such growth differences. Results Here, we developed a computerized image analysis system called Growth Detector (GD), to automatically acquire quantitative and comparative information for yeast colony growth. GD offers great convenience and accuracy over the currently used manual growth measurement method. It distinguishes true yeast colonies in a digital image and provides an accurate coordinate oriented map of the colony areas. Some post-processing calculations are also conducted. Using GD, we successfully detected a genetic linkage between the molecular activity of the plant-derived antifungal compound berberine and gene expression components, among other cellular processes. A novel association for the yeast mek1 gene with DNA damage repair was also identified by GD and confirmed by a plasmid repair assay. The results demonstrate the usefulness of GD for yeast functional genomics research. Conclusion GD offers significant improvement over the manual inspection method to detect relative yeast colony size differences. The speed and accuracy associated with GD makes it an ideal choice for large-scale functional genomics investigations.

Objective We previously identified propionic acid as a microbially-produced volatile organic compound with fungicidal activity against several pathogenic fungi. The purpose of this work is to better understand how propionic acid affects fungi by examining some of the effects of this compound on the yeast cell. Results We show that propionic acid causes a dramatic increase in the uptake of lucifer yellow in yeast cells, which is consistent with enhanced endocytosis. Additionally, using a propidium iodide assay, we show that propionic acid treatment causes a significant increase in the proportion of yeast cells in G1 and a significant decrease in the proportion of cells in G2, suggesting that propionic acid causes a cell cycle arrest in yeast. Finally, we show that the reduction of MTT is attenuated in yeast cells treated with propionic acid, indicating that propionic acid disrupts cellular respiration. Understanding the effects of propionic acid on the yeast cell may aid in assessing the broader utility of this compound.

We present a spectrophotometric-based assay to identify enzymes that degrade commercially available bioplastics. Bioplastics comprise aliphatic polyesters with hydrolysis-susceptible ester bonds and are proposed as a replacement for petroleum-based plastics that accumulate in the environment. Unfortunately, many bioplastics can also persist in environments including seawater and waste centers. Our assay involves an overnight incubation of candidate enzyme(s) with plastic, followed by A610 spectrophotometry using 96-well plates to quantify both a reduction in residual plastic and the liberation of degradation by-products. We use the assay to show that Proteinase K and PLA depolymerase, two enzymes that were previously shown to degrade pure polylactic acid plastic, promote a 20–30% breakdown of commercial bioplastic during overnight incubation. We validate our assay and confirm the degradation potential of these enzymes with commercial bioplastic using established mass-loss and scanning electron microscopy methods. We show how the assay can be used to optimize parameters (temperature, co-factors, etc.) to enhance the enzyme-mediated degradation of bioplastics. The assay endpoint products can be coupled with nuclear magnetic resonance (NMR) or other analytical methods to infer the mode of enzymatic activity. Overall, the screening capacity of the spectrophotometric-based assay was demonstrated to be an accurate method to identify bioplastic-degrading enzymes.

The underlying molecular mechanisms of programmed cell death associated with fungal allorecognition, a form of innate immunity, remain largely unknown. In this study, transcriptome analysis was used to infer mechanisms activated during barrage formation in vic3-incompatible strains of Cryphonectria parasitica, the chestnut blight fungus. Pronounced differential expression occurred in barraging strains of genes involved in mating pheromone (mf2-1, mf2-2), secondary metabolite production, detoxification (including oxidative stress), apoptosis-related, RNA interference, and HET-domain genes. Evidence for secondary metabolite production and reactive oxygen species (ROS) accumulation is supported through UPLC-HRMS analysis and cytological staining, respectively. Differential expression of mating-related genes and HET-domain genes was further examined by RT-qPCR of incompatible interactions involving each of the six vegetative incompatibility (vic) loci in C. parasitica and revealed distinct recognition process networks. We infer that vegetative incompatibility in C. parasitica activates defence reactions that involve secondary metabolism, resulting in increased toxicity of the extra- and intracellular environment. Accumulation of ROS (and other potential toxins) may result in detoxification failure and activation of apoptosis, sporulation, and the expression of associated pheromone genes. The incompatible reaction leaves abundant traces of a process-specific metabolome as conidiation is initiated.

Pseudogymnoascus destructans, the fungal pathogen that causes white-nose syndrome, has killed millions of bats across eastern North America and continues to threaten new bat populations. The spread and persistence of P. destructans has likely been worsened by the ability of this fungus to grow as a saprotroph in the hibernaculum environment. Reducing the environmental growth of P. destructans may improve bat survival. Volatile organic compounds (VOCs) are attractive candidates to target environmental P. destructans, as they can permeate through textured environments that may be difficult to thoroughly contact with other control mechanisms. We tested in hibernaculum sediment the performance of VOCs that were previously shown to inhibit P. destructans growth in agar cultures and examined the inhibition kinetics and specificity of these compounds. Three VOCs, 2-methyl-1-butanol, 2-methyl-1-propanol, and 1-pentanol, were fungicidal towards P. destructans in hibernaculum sediment, fast-acting, and had greater effects against P. destructans than other Pseudogymnoascus species. Our results suggest that use of these VOCs may be considered further as an effective management strategy to reduce the environmental exposure of bats to P. destructans in hibernacula.

Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L50 = 5; N50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.

Nisin is a class I polycyclic bacteriocin produced by the bacterium Lactococcus lactis, which is used extensively as a food additive to inhibit the growth of foodborne Gram-positive bacteria. Nisin also inhibits growth of Gram-negative bacteria when combined with membrane-disrupting chelators such as citric acid. To gain insight into nisin’s mode of action, we analyzed chemical–genetic interactions and identified nisin-sensitive Escherichia coli strains in the Keio library of knockout mutants. The most sensitive mutants fell into two main groups. The first group accords with the previously proposed mode of action based on studies with Gram-positive bacteria, whereby nisin interacts with factors involved in cell wall, membrane, envelope biogenesis. We identified an additional, novel mode of action for nisin based on the second group of sensitive mutants that involves cell cycle and DNA replication, recombination, and repair. Further analyses supported these two distinct modes of action.

Lithium Chloride (LiCl) toxicity, mode of action and cellular responses have been the subject of active investigations over the past decades. In yeast, LiCl treatment is reported to reduce the activity and alters the expression of PGM2, a gene that encodes a phosphoglucomutase involved in sugar metabolism. Reduced activity of phosphoglucomutase in the presence of galactose causes an accumulation of intermediate metabolites of galactose metabolism leading to a number of phenotypes including growth defect. In the current study, we identify two understudied yeast genes, YTA6 and YPR096C that when deleted, cell sensitivity to LiCl is increased when galactose is used as a carbon source. The 5'-UTR of PGM2 mRNA is structured. Using this region, we show that YTA6 and YPR096C influence the translation of PGM2 mRNA.

Context: Plants of the genus Echinacea (Asteraceae) are among the most popular herbal supplements on the market today. Recent studies indicate there are potential new applications and emerging markets for this natural health product (NHP). Objective: This review aims to synthesize recent developments in Echinacea biotechnology and to identify promising applications for these advances in the industry. Methods: A comprehensive survey of peer-reviewed publications was carried out, focusing on Echinacea biotechnology and impacts on phytochemistry. This article primarily covers research findings since 2007 and builds on earlier reviews on the biotechnology of Echinacea. Results: Bioreactors, genetic engineering and controlled biotic or abiotic elicitation have the potential to significantly improve the yield, consistency and overall quality of Echinacea products. Using these technologies, a variety of new applications for Echinacea can be realized, such as the use of seed oil and antimicrobial and immune boosting feed additives for livestock. Conclusions: New applications can take advantage of the well-established popularity of Echinacea as a NHP. Echinacea presents a myriad of potential health benefits, including anti-inflammatory, anxiolytic and antibiotic activities that have yet to be fully translated into new applications. The distinct chemistry and bioactivity of different Echinacea species and organs, moreover, can lead to interesting and diverse commercial opportunities.

Plants of the genus Echinacea have received extensive research attention, yet relatively little is known about the fruit (cypselae) of this economically important genus. The goal of this study was to investigate and compare the cypselae of four species of Echinacea: E. purpurea (L.) Moench, E. angustifolia DC., E. pallida (Nutt.) Nutt., and E. paradoxa (Norton) Britton, with respect to chemistry and germination. The phytochemical content of various fruit fractions was analyzed by high performance liquid chromatography with diode-array detection, identifying several alkylamides and caffeic-acid derivatives of medicinal value in all of the tissues. Alkylamides were most concentrated in the perianth and glands, whereas phenolics were generally enriched within the seed. Germination studies of intact cypselae and de-coated seeds demonstrate that the influence of cypsela coatings on germination depends on the growth environment. Removal of the perianth and pericarp resulted in decreased germination in soil, but significantly improved the germination of seeds of all species when sown in a sterile agar medium. In contrast, a higher percentage of intact cypselae of E. purpurea, E. angustifolia, and E. pallida germinated in soil than in sterile agar. These results suggest an ecophysiological role for phytochemicals in seed germination and will inform the cultivation and breeding of less common Echinacea species as well as the development of new products from currently unused plant material.

Balancing selection has been inferred in diverse organisms for nonself recognition genes, including those involved in immunity, mating compatibility, and vegetative incompatibility. Although selective forces maintaining polymorphisms are known for genes involved in immunity and mating, mechanisms of balancing selection for vegetative incompatibility genes in fungi are being debated. We hypothesized that allorecognition and its consequent inhibition of virus transmission contribute to the maintenance of polymorphisms in vegetative incompatibility loci (vic) in the chestnut blight fungus, Cryphonectria parasitica. Balancing selection was demonstrated at two loci, vic2 and vic6, by trans-species polymorphisms in C. parasitica, C. radicalis, and C. japonica and signatures of positive selection in gene sequences. In addition, more than half (31 of 54) of allele frequency estimates at six vic loci in nine field populations of C. parasitica from Asia and the eastern US were not significantly different from 0.5, as expected at equilibrium for two alleles per locus under balancing selection. At three vic loci, deviations from 0.5 were predicted based on the effects of heteroallelism on virus transmission. Twenty-five of 27 allele frequency estimates were greater than or equal to 0.5 for the allele that confers significantly stronger inhibition of virus transmission at three loci with asymmetric transmission. These results are consistent with the allorecognition hypothesis that vegetative incompatibility genes are under selection because of their role in reducing infection by viruses.

Vegetative incompatibility (VI) is a form of non-self allorecognition in filamentous fungi that restricts conspecific hyphal fusion and the formation of heterokaryons. In the chestnut pathogenic fungus, Cryphonectria parasitica, VI is controlled by six vic loci and has been of particular interest because it impedes the spread of hypoviruses and thus biocontrol strategies. We use nuclear magnetic resonance and high-resolution mass spectrometry to characterize alterations in the metabolome of C. parasitica over an eight-day time course of vic3 incompatibility. Our findings support transcriptomic data that indicated remodeling of secondary metabolite profiles occurs during vic3 -associated VI. VI-associated secondary metabolites include novel forms of calbistrin, decumbenone B, a sulfoxygenated farnesyl S-cysteine analog, lysophosphatidylcholines, and an as-yet unidentified group of lipid disaccharides. The farnesyl S-cysteine analog is structurally similar to pheromones predicted to be produced during VI and is here named 'crypheromonin'. Mass features associated with C. parasitica secondary metabolites skyrin, rugulosin and cryphonectric acid were also detected but were not VI specific. Partitioning of VI-associated secondary metabolites was observed, with crypheromonins and most calbistrins accumulating in the growth medium over time, whereas lysophosphatidylcholines, lipid disaccharide-associated mass features and other calbistrin-associated mass features peaked at distinct time points in the mycelium. Secondary metabolite biosynthetic gene clusters and potential biological roles associated with the detected secondary metabolites are discussed.

The fate, toxicity and bioaccumulation of copper oxide nanoparticles (nCuO) was investigated in soil, with and without biosolids amendment, through chronic exposures using the earthworm, Eisenia andrei, and the collembolan, Folsomia candida. The effects of copper sulphate (CuSO4) were included so as to compare the behavior of nCuO to a readily soluble counterpart. The fate of nCuO was evaluated through characterization of dissolved and nano-particulate fractions (via single particle ICP-MS) as well as extractable Cu2+ throughout the duration of select tests. Neither Cu form was particularly toxic to F. candida, but effects on E. andrei reproduction were significant in all treatments (IC50 range: 98 – 149 mg Cu kg−1 dry soil). There were no significant differences in toxicity between the Cu forms, nor in extractable Cu2+ activities, indicative that particle dissolution within the soil and, subsequent activity of Cu2+ was likely the primary mode of toxicity in the nCuO exposures. The presence of biosolids did not significantly alter toxicity of nCuO, but did affect Cu2+ activity over time. Bioaccumulation of total Cu in E. andrei when exposed to nCuO (kinetic bioaccumulation factor (BAFk): 0.80 with biosolids and 0.81 without) was lower than exposure to CuSO4 (BAFk: 2.31 with biosolids and 1.12 without). Enhanced dark-field hyperspectral imaging showed accumulation of nCuO along the epidermis and gut of E. andrei, with trace amounts observed in muscle and chloragogenous tissue, providing evidence of nCuO translocation within the organism. The present study demonstrates that the current risk assessment approach for trace metals in the environment, based on substance solubility and bioavailability of the dissolved free ion, are applicable for nCuO exposure to soil invertebrates, but that the rate of particle dissolution in different soil environments is an important factor for consideration.

Nisin is a class I polycyclic bacteriocin produced by the bacterium

Fusarium head blight caused by the fungal pathogen Fusarium graminearum is a devastating disease of cereal crops worldwide, with limited effective chemical treatments available. Here we show that the natural alkaloid compound antofine can inhibit fusarium head blight in wheat. Using yeast genomic screening, we identified the TOR pathway component RRD2 as a target of antofine that is also required for F. graminearum pathogenicity., Antofine, a phenanthroindolizidine alkaloid, is a bioactive natural product isolated from milkweeds that exhibits numerous biological activities, including anticancer, antimicrobial, antiviral, and anti-inflammatory properties. However, the direct targets and mode of action of antofine have not been determined. In this report, we show that antofine displays antifungal properties against the phytopathogen Fusarium graminearum, the cause of Fusarium head blight disease (FHB). FHB does devastating damage to agriculture, causing billions of dollars in economic losses annually. We therefore sought to understand the mode of action of antofine in F. graminearum using insights from yeast chemical genomic screens. We used haploinsufficiency profiling (HIP) to identify putative targets of antofine in yeast and identified three candidate targets, two of which had homologs in F. graminearum. The Fusarium homologues of two targets, glutamate dehydrogenase (FgGDH) and resistance to rapamycin deletion 2 (FgRRD2), can bind antofine. Of the two genes, only the Fgrrd2 knockout displayed a loss of virulence in wheat, indicating that RRD2 is an antivirulence target of antofine in F. graminearum. Mechanistically, we demonstrate that antofine disrupts the interaction between FgRRD2 and FgTap42, which is part of the Tap42-phosphatase complex in the target of rapamycin (TOR) signaling pathway, a central regulator of cell growth in eukaryotes and a pathway of extensive study for controlling numerous pathologies.

The masked birch caterpillar, Drepana arcuata Walker (Lepidoptera: Drepanidae), and other Drepanoidea (Lepidoptera) species are excellent organisms for investigating the function and evolution of vibratory communication and sociality in caterpillars. We present a de novo assembled draft genome and functional annotation for D. arcuata, using a combination of short and long sequencing reads generated by Illumina HiSeq X and Oxford Nanopore Technologies (ONT) MinION sequencing platforms, respectively. A total of 460,694,612 150bp paired-end Illumina and 395,890 ONT raw reads were assembled into 11,493 scaffolds spanning a genome size of 270.5Mb. The resulting D. arcuata genome has a GC content of 38.79%, repeat content of 8.26%, is 86.5% complete based on Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment, and comprises 10,398 predicted protein-coding genes. These data represent the first genomic resources for the lepidopteran superfamily Drepanoidea. Although the order Lepidoptera comprises numerous ecologically and economically important species, assembled genomes and annotations are available for < 1% of the total species. These data can be further utilized for research on Lepidoptera genomics as well as on the function and evolution of vibratory communication and sociality in larval insects.

For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5&prime, Untranslated region (5&prime, UTR) of PGM2 mRNA in yeast.

The masked birch caterpillar, Drepana arcuata, provides an excellent opportunity to study mechanisms mediating developmental changes in social behaviour. Larvae transition from being social to solitary during the 3rd instar, concomitant with shifts in their use of acoustic communication. In this study we characterize the transcriptome of D. arcuata to initiate sociogenomic research of this lepidopteran insect. We assembled and annotated the combined larval transcriptome of "social" early and "solitary" late instars using next generation Illumina sequencing, and used this transcriptome to conduct differential gene expression analysis of the two behavioural phenotypes. A total of 211,012,294 reads generated by RNA sequencing were assembled into 231,348 transcripts and 116,079 unigenes for the functional annotation of the transcriptome. Expression analysis revealed 3300 transcripts that were differentially expressed between early and late instars, with a large proportion associated with development and metabolic processes. We independently validated differential expression patterns of selected transcripts using RT-qPCR. The expression profiles of social and solitary larvae revealed differentially expressed transcripts coding for gene products that have been previously reported to influence social behaviour in other insects (e.g. cGMP- and cAMP- dependent kinases, and bioamine receptors). This study provides the first transcriptomic resources for a lepidopteran species belonging to the superfamily Drepanoidea, and gives insight into genetic factors mediating grouping behaviour in insects.

In the late 1980s, Smith, Bruhn, and Anderson discovered a genetic individual of the fungus Armillaria gallica that extended over 37 hectares of forest floor and encompassed hundreds of tree root systems in Northern Michigan. Based on observed growth rates, the individual was estimated to be at least 1500 years old with a mass of 100,000 kg. The conclusion was that the Michigan individual of A. gallica was among the largest and oldest organisms on earth, a remarkable claim given that Armillaria is essentially a microorganism existing largely as microscopic hyphae embedded in their substrate. Nearly three decades on, we returned to the site of the large Michigan individual with the tools of whole-genome sequencing. Here, we show (a) that the large individual is still alive on its original site and (b) that mutation has occurred within the somatic cells of the large individual, reflecting its historical pattern of growth from a single point. The overall rate of mutation, however, was extremely low. On the spectrum of mutability in somatic cells, Armillaria occupies the extreme of stability, opposite the extreme of instability as typified by cancer.

Engineered nanomaterials (ENMs) are increasingly incorporated into a variety of commercial applications and consumer products; however, ENMs may possess cytotoxic properties due to their small size. This study assessed the effects of two commonly used ENMs, zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs), in the model eukaryote Saccharomyces cerevisiae. A collection of ≈4600 S. cerevisiae deletion mutant strains was used to deduce the genes, whose absence makes S. cerevisiae more prone to the cytotoxic effects of ZnONPs or AgNPs. We demonstrate that S. cerevisiae strains that lack genes involved in transmembrane and membrane transport, cellular ion homeostasis, and cell wall organization or biogenesis exhibited the highest sensitivity to ZnONPs. In contrast, strains that lack genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport exhibited the highest sensitivity to AgNPs. Secondary assays confirmed that ZnONPs affected cell wall function and integrity, whereas AgNPs exposure decreased transcription, reduced endocytosis, and led to a dysfunctional electron transport system. This study supports the use of S. cerevisiae Gene Deletion Array as an effective high-throughput technique to determine cellular targets of ENM toxicity.

Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside (1) and new saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-β-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 μM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 μM).

Mobile young under parental care have a high potential for intermixing with other broods, which potentially increases the costs to the foster parents. Here, we examined for the first time the genetic composition of wild-caught broods of the convict cichlid (Amatitlania siquia), a socially monogamous biparental fish, for evidence of brood mixing and adoption. Our microsatellite genotyping data revealed that 79% of broods contained adopted young. Moreover, 25% of broods contained adopted sibsets likely arising from extra-pair matings, a phenomenon hitherto not documented for this species. Overall, adopted foreign fry and host fry in mixed broods were generally different in body length, as would be expected if they have different parents. However, fry from possible extra-pair matings were similar in body length to host fry, suggesting that they are of similar age. Our results are important because they reveal a very high prevalence and degree of brood mixing, and indicate that social monogamy does not necessarily lead to genetic monogamy in the convict cichlid in nature. These findings raise questions about potential brood-mixing mechanisms and the reproductive ecology (especially opportunities for polygamy in nature) of this important model species in the study of animal behaviour.

The presence of acetic acid during industrial alcohol fermentation reduces the yield of fermentation by imposing additional stress on the yeast cells. The biology of cellular responses to stress has been a subject of vigorous investigations. Although much has been learned, details of some of these responses remain poorly understood. Members of heat shock chaperone HSP proteins have been linked to acetic acid and heat shock stress responses in yeast. Both acetic acid and heat shock have been identified to trigger different cellular responses including reduction of global protein synthesis and induction of programmed cell death. Yeast HSC82 and HSP82 code for two important heat shock proteins that together account for 1-2% of total cellular proteins. Both proteins have been linked to responses to acetic acid and heat shock. In contrast to the overall rate of protein synthesis which is reduced, the expression of HSC82 and HSP82 is induced in response to acetic acid stress. In the current study we identified two yeast genes DOM34 and RPL36A that are linked to acetic acid and heat shock sensitivity. We investigated the influence of these genes on the expression of HSP proteins. Our observations suggest that Dom34 and RPL36A influence translation in a CAP-independent manner., This work was funded by the Natural Sciences and Engineering Research Council of Canada, NSERC., info:eu-repo/semantics/publishedVersion

The capacity of two soil fungi, Trichoderma koningii and Penicillium janthinellum, to oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and store intracellular lipids during growth is unknown. This article reports for the first time the metabolism of decanoic acid (DA, C10:0), undecanoic acid (UDA, n-C11:0), a mixture of the acids (UDA+DA) and a mixture of UDA+ potato dextrose broth (PDB) by T. koningii and P. janthinellum and their mixed culture. A control PDB complex substrate was used as a substrate control treatment. The fungal cultures were assayed for their capacity to: (1) oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and (2) store lipids intracellularly during growth. On all four fatty acid substrates, the mixed T. koningii and P. janthinellum culture produced more biomass and CO2 than the individual fungal cultures. Per 150 mL culture, the mixed species culture grown on UDA+PDB and on PDB alone produced the most biomass (7,567 mg and 11,425 mg, respectively). When grown in DA, the mixed species culture produced the least amount of biomass (6,400 mg), a quantity that was lower than those obtained in UDA (7,550 mg) or UDA+DA (7,270 mg). Amounts of CO2 produced ranged from 210 mg under DA to 618 mg under PDB, and these amounts were highly correlated with biomass (r(2) = 0.99). Fluorescence microscopy of stained lipids in the mixed fungal cell cultures growing during the exponential phase demonstrated larger fungal cells and higher accumulation of lipids in membranes and storage bodies than those observed during the lag and stationary phases. T. koningii and P. janthinellum grown on n-C10:0 and n-C11:0 fatty acids produced lower amounts of biomass and CO2, but stored higher amounts of intracellular lipids, than when grown on PDB alone.

Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling.

The antifungal mode of action of thymol was investigated by a chemical-genetic profile analysis. Growth of each of ~4700 haploid Saccharomyces cerevisiae gene deletion mutants was monitored on medium with a subinhibitory concentration (50 μg/ml) of thymol and compared to growth on non-thymol control medium. This analysis revealed that, of the 76 deletion mutants with the greatest degree of susceptibility to thymol, 29% had deletions in genes involved in telomere length maintenance. A telomere restriction fragment (TRF) length assay showed that yeast exposed to a subinhibitory concentration of thymol for 15 days had telomere size reductions of 13-20% compared to non-thymol controls. By accelerating telomere shortening, thymol may increase the rate of cell senescence and apoptosis. Furthermore, real-time RT-PCR analysis revealed approximately two-fold reductions in EST2 mRNA but no change in TLC1 RNA in thymol-treated S. cerevisiae relative to untreated cells. EST2 encodes the essential reverse transcriptase subunit of telomerase that uses TLC1 RNA as a template during addition of TG(1-3) repeats to maintain telomere ends. This study provides compelling evidence that a primary mode of thymol antifungal activity is through inhibition of transcription of EST2 and thus telomerase activity.

The antimicrobial activity of chitosan has been acknowledged for more than 30 years and yet its mode-of-action remains ambiguous. We analyzed chemical-genetic interactions of low-molecular weight chitosan using a collection of ≈ 4600 S. cerevisiae deletion mutants and found that 31% of the 107 mutants most sensitive to chitosan had deletions of genes related primarily to functions involving protein synthesis. Disruption of protein synthesis by chitosan was substantiated by an in vivo β-galactosidase expression assay suggesting that this is a primary mode of antifungal action. Analysis of the yeast gene deletion array and secondary assays also indicate that chitosan has a minor membrane disruption effect - a leading model of chitosan antimicrobial activity.

Type I ribonucleotide reductases (RNRs) are conserved across diverse taxa and are essential for the conversion of RNA into DNA precursors. In Neurospora crassa, the large subunit of RNR (UN-24) is unusual in that it also has a nonself recognition function, whereby coexpression of Oak Ridge (OR) and Panama (PA) alleles of un-24 in the same cell leads to growth inhibition and cell death. We show that coexpressing these incompatible alleles of un-24 in N. crassa results in a high molecular weight UN-24 protein complex. A 63-amino-acid portion of the C terminus was sufficient for un-24PA incompatibility activity. Redox active cysteines that are conserved in type I RNRs and essential for their catalytic function were found to be required for incompatibility activity of both UN-24OR and UN-24PA. Our results suggest a plausible model of un-24 incompatibility activity in which the formation of a complex between the incompatible RNR proteins is potentiated by intermolecular disulfide bond formation.

Pseudogymnoascus destructans, the fungus that causes white-nose syndrome in hibernating bats, has spread across eastern North America over the past decade and decimated bat populations. The saprotrophic growth of P. destructans may help to perpetuate the white-nose syndrome epidemic, and recent model predictions suggest that sufficiently reducing the environmental growth of P. destructans could help mitigate or prevent white-nose syndrome-associated bat colony collapse. In this study, we screened 301 microbes from diverse environmental samples for their ability to inhibit the growth of P. destructans. We identified 145 antagonistic isolates, 53 of which completely or nearly completely inhibited the growth of P. destructans in co-culture. Further analysis of our best antagonists indicated that these microbes have different modes of action and may have some specificity in inhibiting P. destructans. The results suggest that naturally-occurring microbes and/or their metabolites may be considered further as candidates to ameliorate bat colony collapse due to P. destructans.

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.

The neotropical convict cichlid fish (Amatitlania siquia) is an important model species for the study of animal behaviour. It is therefore surprising that no genetic markers are available for this species. Here, we have optimised four poly- morphic microsatellite loci for use with this fish, where each locus comprises between 9 and 28 alleles. We demonstrate how these markers can be used to genotype fry (young) and for the analyses of genetic structure and prevalence of interfamilial mixing within biparental broods of wild convict cichlids. These microsatellites will facilitate future research, hitherto not possible, on the molecular, behavioural and evolutionary ecology of the convict cichlid and possibly other closely related species.

Background:SchS21 and SchS34 are proteins from Stachybotrys chartarum sensu latto that are antigenic in goats, mice and humans. Monoclonal antibodies to these proteins react with spores of S. chartarum and S. chlorohalonata but do not cross-react with a diverse taxonomic and ecological array of other fungi. Methods: Based on partial sequences of the 21- and 34-kDa proteins, obtained from tandem mass spectra and Edman degradation, degenerate primers were designed for touchdown PCR and the resulting amplicons were sequenced. Subsequently, inverse-PCR was used to obtain genomic DNA sequences encoding SchS21 and SchS34. RT-PCR products were sequenced to predict the mature protein sequences of SchS21 and SchS34. Based on the speculation that SchS21 protein was a DNase, the enzymatic properties were investigated. Results: Sequences of 435 and 666 bp in length were obtained from SchS21 and SchS34 cDNAs. The SchS21 open reading frame encodes a mature protein of 144 amino acids, while that of SchS34 is 221 amino acids in length. SchS21 is a secretory, alkaline, Mg-dependent exodeoxyribonuclease, while SchS34 is a secretory protein of unknown function. His-tagged forms of the mature SchS21 and SchS34 proteins were separately overexpressed in Escherichia coli and purified using Ni-NTA columns (0.5 mg/l yield). Conclusions: Based on Western blots, the expressed proteins were similar in molecular weight and bound to the respective monoclonal antibodies to SchS21 and SchS34 proteins from S. chartarum. Interactions with human sera IgE confirmed the expressed forms of SchS21 and SchS34 as naturally occurring allergens.

In addition to widespread use in reducing the symptoms of colds and flu, Echinacea is traditionally employed to treat fungal and bacterial infections. However, to date the mechanism of antimicrobial activity of Echinacea extracts remains unclear. We utilized a set of#x223C;4,600 viable gene deletion mutants of Saccharomyces cerevisiae to identify mutations that increase sensitivity to Echinacea. Thus, a set of chemical-genetic profiles for 16 different Echinacea treatments was generated, from which a consensus set of 23 Echinacea-sensitive mutants was identified. Of the 23 mutants, only 16 have a reported function. Ten of these 16 are involved in cell wall integrity/structure suggesting that a target for Echinacea is the fungal cell wall. Follow-up analyses revealed an increase in sonication-associated cell death in the yeasts S. cerevisiae and Cryptococcus neoformans after Echinacea extract treatments. Furthermore, fluorescence microscopy showed that Echinacea-treated S. cerevisiae was significantly more prone to cell wall damage than non-treated cells. This study further demonstrates the potential of gene deletion arrays to understand natural product antifungal mode of action and provides compelling evidence that the fungal cell wall is a target of Echinacea extracts and may thus explain the utility of this phytomedicine in treating mycoses.

This study examines decontamination processes that were developed on an emergency basis to eliminate Bacillus anthracis spores from deliberately contaminated buildings. The recommended steps include a survey with sampling, the removal of sensitive items, and HEPA vacuuming of all readily available surfaces, followed by biocide treatment and subsequent analyses for viable cells. There are several analytical challenges posed by this approach. These include the ability to discriminate the added strain from naturally occurring resident microbes, determining detection limits for anthrax spores in settled dusts, and detecting viable but nonculturable spores. There are also logistical issues relating to the various skill sets required from investigation to reconstruction. In the present study, a model office was constructed, and a strain of Bacillus pumilus was isolated from the carpet and reintroduced to the office in excess. The abundance of the B. pumilus strain was monitored in settled dust using a strain-specific, quantitative polymerase chain reaction (QPCR)-based detection method following repeated HEPA vacuum cleanings. The QPCR method had a limit of detection corresponding toor = 10(2) colony forming units per gram of settled dust. QPCR results were compared with measures of dust recoveries and fungal glucan and endotoxin levels in the dust samples. The largest fraction (ca. 81%) of added spores was recovered during the first HEPA cleaning. Subsequent cleanings resulted in incrementally lower recoveries, with removal of 93% of the initial inoculum by the third HEPA vacuuming. HEPA vacuuming prior to removal of items such as office contents and furnishings would result in much less resuspension of dust and limiting the extent of contamination. This approach also ensures that residual contaminants are as low as can be reasonably achieved.

Real-time fluorescence detection systems were adapted to identify DNA adducts formed by photogenotoxic phytochemicals. Two assays were developed: the first was based on quantitative polymerase chain reaction (qPCR) while the second used thermal denaturation and renaturation (D-R). Both assays employed yeast DNA, the fluorescent dye SYBR Green and a real-time PCR thermocycler. The furanocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), psoralen, angelicin and imperatorin, and the furanochrome khellin, were tested for adduct forming ability with up to 2 h of UVA light exposure (λ = 320–400 nm). The known bifunctional compounds, 8-MOP, 5-MOP and psoralen, were inferred to form biadducts here based on both D-R and qPCR assays, as expected from previous research. The known monofunctional compound angelicin was used as a negative control and did not form biadducts based on either assay. Two compounds of unknown functional specificity, imperatorin and khellin, were determined to be positive and negative for biadduct activity, respectively. Detection of biadducts with 8-MOP, 5-MOP, psoralen and imperatorin, but not angelicin or khellin, was further verified by temperature gradient gel electrophoresis. The fluorescence methods improve and expand upon existing assays to monitor DNA adducts. Copyright © 2008 John Wiley & Sons, Ltd.

We tested extracts of Vincetoxicum rossicum for inhibition of bacterial and fungal growth and for anti-insect activities that may account for the invasive characteristics of this introduced species in North America. Bioassay-guided fractionation was used to identify (� )-antofine as the principle inhibitor of bacteria and fungi in root extracts. This compound had especially pronounced antifungal activity, inhibiting thegrowth of diverse taxa that includeyeast-like andfilamentous fungi and, notably, broad-host-range plant pathogens. A second compound(s), that is as yet uncharacterized but distinct from (� )-antofine, was detected as having antifeedant activity against a larval hymenopteran, rose sawfly (Allantus cinctus), and toxicity to two larval lepidopterans, the masked birch caterpillar (Drepana arcuata) and the European corn borer (Ostrinia nubilalis). That V. rossicum contains potent inhibitors of plant pathogenic fungi, diverse bacteria, and herbivorous insects likely contributes to its success as an invasive species. 2008 Elsevier Ltd. All rights reserved.

Bioassay-guided fractionation of Chimaphila umbellata (L.) W. Bart (Pyrolaceae) ethanol extracts led to the identification of 2,7-dimethyl-1,4-naphthoquinone (chimaphilin) as the principal antifungal component. The structure of chimaphilin was confirmed by 1H and 13C NMR spectroscopy. The antifungal activity of chimaphilin was evaluated using the microdilution method with Saccharomyces cerevisiae (0.05mg/mL) and the dandruff-associated fungi Malassezia globosa (0.39mg/mL) and Malassezia restricta (0.55mg/mL). Pronounced antioxidant activity of C. umbellata crude extract was also identified using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, suggesting this phytomedicine has an antioxidant function in wound healing. A chemical-genetic profile was completed with chimaphilin using approximately 4700 S. cerevisiae gene deletion mutants. Cellular roles of deleted genes in the most susceptible mutants and secondary assays indicate that the targets for chimaphilin include pathways involved in cell wall biogenesis and transcription.

Identification of novel antifungals is needed, and plant-derived compounds offer important leads in this respect. This article initiates a compendium of secondary assays for testing hypotheses on mode of action of plant-derived compounds. Three assays were developed to evaluate compounds that are hypothesized to effect cell wall function, transcription, and oxidative stress. A hypothesized perturbation on cell wall integrity by chimaphilin and Echinacea. root extracts was supported using sonication and heat shock assays. Heat shock treatments reduced the number of viable cells recovered when yeast was grown in the presence of subinhibitory concentrations of chimaphilin. A reduction in cell numbers was also observed with both sonication-based and heat shock–based assays when yeast was grown in the presence of Echinacea purpurea. (L.) Moench (Asteraceae) extracts. The hypothesis that chimaphilin impedes mRNA transcription processes was also supported by a β-galactosidase reporter gene assay. The ant...

Antifungal activity is positively correlated to furanocoumarin content in extracts of the traditional phytomedicine northern prickly ash (Zanthoxylum americanum Mill. [Rutaceae]). The specificity of these furanocoumarins in inhibiting replication of DNA was investigated with reference to significant base composition differences between fungal and mammalian mitochondrial DNA. We developed a polymerase chain reaction-based assay to investigate whether (1) furanocoumarins inhibit DNA polymerization and (2) distinct furanocoumarins specifically inhibit DNA replication depending on base composition. Specific inhibition of DNA polymerization by 5-methoxypsoralen and psoralen through high-adenine and thymine (AT) (84.3%) and low-AT (51.9%) DNA, respectively, suggests that furanocoumarins inhibit replicative functions of genomes or of regions within the genome that differ in base composition. Greater overall inhibition of DNA polymerization by Z. americanum husk extracts than with single or mixed furanocoumarins suggests that inhibitory compounds in addition to the major furanocoumarins are present in Z. americanum.

Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside (1) and new saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-β-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 μM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 μM).

The Marcgraviaceae is a neotropical plant family of lianas and shrubs distributed throughout Central and South America, mainly in evergreen and semi-evergreen tropical forests. In this study, extracts of 12 Marcgraviaceae species were assessed for inhibition of bacterial quorum sensing (QS) in Chromobacterium violaceum, bacterial biofilm formation in Pseudomonas aeruginosa PA14, and fungal growth in Saccharomyces cerevisiae. Six species showed promising inhibitory activities in at least one of the three bioassays (Marcgravia nervosa Triana & Planch., Marcgravia polyantha Delp., Marcgravia schippii Standl., Marcgraviastrum subsessile (Benth.) Bedell, Schwartzia brasiliensis (Choisy) Bedell ex Gir-Cañas, and Schwartzia costaricensis (Gilg.) Bedell). Analyses of the crude extract of the leaves of Marcgravia nervosa using HPLC–APCI–MS showed the presence of five pentacyclic triterpenes: ursolic acid (2), betulinic acid (3), α–amyrin (4), β–amyrin (5), lupeol (6). Bioassay-guided fractionation of this plant resulted in the isolation and identification of 2-methoxy-1,4-naphthoquinone (1) as the active principle with a minimum inhibitory concentration (MIC) of 85–100 μmol·L−1 against Saccharomyces cerevisiae BY4741 (haploid) and BY4743 (diploid). Las Marcgraviaceae son una familia de plantas neotropicales de lianas y arbustos distribuidas a lo largo de América Central y del Sur, principalmente en bosques tropicales perennifolios y semiperennifolios. En este estudio, se evaluaron extractos de 12 especies de Marcgraviaceae para inhibir la detección de quórum bacteriano (QS) en Chromobacterium violaceum, la formación de biopelículas bacterianas en Pseudomonas aeruginosa PA14 y el crecimiento de hongos en Saccharomyces cerevisiae. Seis especies mostraron actividades inhibitorias prometedoras en al menos uno de los tres bioensayos (Marcgravia nervosa Triana & Planch., Marcgravia polyantha Delp., Marcgravia schippii Standl., Marcgraviastrum subsessile (Benth.) Bedell, Schwartzia brasiliensis (Choisy) Bedell ex Gir-Cañas , y Schwartzia costaricensis (Gilg.) Bedell). Los análisis del extracto crudo de las hojas de Marcgravia nervosa mediante HPLC-APCI-MS mostraron la presencia de cinco triterpenos pentacíclicos: ácido ursólico (2), ácido betulínico (3), α-amirina (4), β-amirina (5) , lupeol (6). El fraccionamiento guiado por bioensayo de esta planta resultó en el aislamiento e identificación de 2-metoxi-1,4-naftoquinona (1) como principio activo con una concentración inhibitoria mínima (MIC) de 85–100 μmol·L−1 contra Saccharomyces cerevisiae BY4741 (haploide) y BY4743 (diploide). Universidad Nacional, Costa Rica Escuela de Química

The damselfly, Nehalennia irene (Hagen), has two distinct female colour morphs. Individuals of one morph have male-like colouration and pattern (androchromes), whereas gynochromes are different from males and androchromes in these respects. In several damselflies, such female-limited polychromatism is attributable to a single genetic locus. We developed six polymorphic genetic markers, which were codominant, to test for genetic differentiation in N. irene, collected from two sites located 8 km from one another in eastern Ontario, Canada. Based on three censuses spanning a 10 year period (1992-2001), morph ratios differed consistently and significantly between these two sites. However, subpopulations at these sites were not genetically differentiated with respect to the putatively neutral markers. Our results suggest that site differences in morph ratios of female N. irene cannot be explained by genetic drift, but are consistent with spatially variable selection operating on different morphs, perhaps mediated by male density. Alternatively, morph type may be a plastic trait and cues for induction may differ between sites.