Spectrophotometric-Based Assay to Quantify Relative Enzyme-Mediated Degradation of Commercially Available Bioplastics.

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.

Octopamine receptor gene influences social grouping in the masked birch caterpillar.

OBJECTIVE: Group-living plays a key role in the success of many insects, but the mechanisms underlying group formation and maintenance are poorly understood. Here we use the masked birch caterpillar, Drepana arcuata, to explore genetic influences on social grouping. These larvae predictably transition from living in social groups to living solitarily during the 3rd instar of development. Our previous study showed a notable shift in the D. arcuata transcriptome that correlates with the transition from grouping to solitary behavior. We noted that one differentially regulated gene, octopamine receptor gene (DaOAR), is a prominent 'social' gene in other insect species, prompting us to test the hypothesis that DaOAR influences grouping behavior in D. arcuata. This was done using RNA interference (RNAi) methods by feeding second instar larvae synthetic dsRNAs. RESULTS: RT-qPCR analysis confirmed a significant reduction in DaOAR transcript abundance in dsRNA-fed larvae compared to controls. Behavioral trials showed that caterpillars with reduced transcript abundance of DaOAR remained solitary throughout the observation period compared to controls. These results provide evidence that regulation of the octopamine receptor gene influences social grouping in D. arcuata, and that specifically, a decrease in octopamine receptor expression triggers the larval transition from social to solitary.

A metabolomic study of vegetative incompatibility in Cryphonectria parasitica.

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.

Propionic acid disrupts endocytosis, cell cycle, and cellular respiration in yeast.

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.

Transcriptome analysis implicates secondary metabolite production, redox reactions, and programmed cell death during allorecognition in Cryphonectria parasitica.

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.

Volatile organic compounds kill the white-nose syndrome fungus, Pseudogymnoascus destructans, in hibernaculum sediment.

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.

Transcriptome analysis of a social caterpillar, Drepana arcuata: De novo assembly, functional annotation and developmental analysis.

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.

Mode of action of nisin on Escherichia coli.

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.

Echinacea biotechnology: advances, commercialization and future considerations.

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.

Zinc oxide and silver nanoparticles toxicity in the baker's yeast, Saccharomyces cerevisiae.

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.

Balancing selection at nonself recognition loci in the chestnut blight fungus, Cryphonectria parasitica, demonstrated by trans-species polymorphisms, positive selection, and even allele frequencies.

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.

Clonal evolution and genome stability in a 2500-year-old fungal individual.

Individuals of the basidiomycete fungus Armillaria are well known for their ability to spread from woody substrate to substrate on the forest floor through the growth of rhizomorphs. Here, we made 248 collections of A. gallica in one locality in Michigan's Upper Peninsula. To identify individuals, we genotyped collections with molecular markers and somatic compatibility testing. We found several different individuals in proximity to one another, but one genetic individual stood out as exceptionally large, covering hundreds of tree root systems over approximately 75 hectares of the forest floor. Based on observed growth rates of the fungus, we estimate the minimum age of the large individual as 2500 years. With whole-genome sequencing and variant discovery, we also found that mutation had occurred within the somatic cells of the individual, reflecting its historical pattern of growth from a single point. The overall rate of mutation over the 90 mb genome, however, was extremely low. This same individual was first discovered in the late 1980s, but its full spatial extent and internal mutation dynamic was unknown at that time. The large individual of A. gallica has been remarkably resistant to genomic change as it has persisted in place.

The sensitivity of the yeast, Saccharomyces cerevisiae, to acetic acid is influenced by DOM34 and RPL36A.

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.

Microbial inhibitors of the fungus Pseudogymnoascus destructans, the causal agent of white-nose syndrome in bats.

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.

Antifungal Saponins from the Maya Medicinal Plant Cestrum schlechtendahlii G. Don (Solanaceae).

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)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranoside (1) and new saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-ß-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).

Metabolism of n-C10:0 and n-C11:0 fatty acids by Trichoderma koningii, Penicillium janthinellum and their mixed culture: I. Biomass and CO2 production, and allocation of intracellular lipids.

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.

Elimination of bioweapons agents from forensic samples during extraction of human DNA.

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.

Antimicrobial and P450 inhibitory properties of common functional foods.

PURPOSE: To study the effect of functional foods on human cytochrome P450 (CYP) and the gut bacterial microflora that may potentially affect drug metabolism and ultimately affect human health and wellness. METHODS: This study examined a variety of food plants from the Apiaceae, Fabaceae, and Lamiaceae families for their inhibitory potential on cytochrome 2D6-, 3A4-, 3A5-, and 3A7-mediated metabolism. The antimicrobial effects of these samples were also investigated with 7 selected bacterial surrogate species to determine potential effects on the gut microflora. RESULTS: The highest CYP inhibitory activities, based upon visual examination, were observed from extracts of celery seed, cumin, fennel seed, basil, oregano, and rosemary belonging to the Apiaceae and Lamiaceae families, respectively. Likewise, the strongest antimicrobial activities were also observed in the Apiaceae and Lamiaceae. No significant antimicrobial and CYP inhibition was observed in the Fabaceae extracts. CONCLUSION: Results demonstrated the possible risk of food-drug interactions from spice and herb plants may affect drug disposition and safety.

Experimental evolution of bet hedging under manipulated environmental uncertainty in Neurospora crassa.

All organisms are faced with environmental uncertainty. Bet-hedging theory expects unpredictable selection to result in the evolution of traits that maximize the geometric-mean fitness even though such traits appear to be detrimental over the shorter term. Despite the centrality of fitness measures to evolutionary analysis, no direct test of the geometric-mean fitness principle exists. Here, we directly distinguish between predictions of competing fitness maximization principles by testing Cohen's 1966 classic bet-hedging model using the fungus Neurospora crassa. The simple prediction is that propagule dormancy will evolve in proportion to the frequency of 'bad' years, whereas the prediction of the alternative arithmetic-mean principle is the evolution of zero dormancy as long as the expectation of a bad year is less than 0.5. Ascospore dormancy fraction in N. crassa was allowed to evolve under five experimental selection regimes that differed in the frequency of unpredictable 'bad years'. Results were consistent with bet-hedging theory: final dormancy fraction in 12 genetic lineages across 88 independently evolving samples was proportional to the frequency of bad years, and evolved both upwards and downwards as predicted from a range of starting dormancy fractions. These findings suggest that selection results in adaptation to variable rather than to expected environments.

A global investigation of gene deletion strains that affect premature stop codon bypass in yeast, Saccharomyces cerevisiae.

Protein biosynthesis is an orderly process that requires a balance between rate and accuracy. To produce a functional product, the fidelity of this process has to be maintained from start to finish. In order to systematically identify genes that affect stop codon bypass, three expression plasmids, pUKC817, pUKC818 and pUKC819, were integrated into the yeast non-essential loss-of-function gene array (5000 strains). These plasmids contain three different premature stop codons (UAA, UGA and UAG, respectively) within the LacZ expression cassette. A fourth plasmid, pUKC815 that carries the native LacZ gene was used as a control. Transformed strains were subjected to large-scale ß-galactosidase lift assay analysis to evaluate production of ß-galactosidase for each gene deletion strain. In this way 84 potential candidate genes that affect stop codon bypass were identified. Three candidate genes, OLA1, BSC2, and YNL040W, were further investigated, and were found to be important for cytoplasmic protein biosynthesis.