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Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungusCaecomyces churrovisand the methanogenMethanobacterium bryantii(not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated inC. churrovisacross a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome ofC. churroviswas obtained and annotated, which is the first sequenced genome of a non-rhizoid forming anaerobic fungus.C. churrovispossess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative toC. churrovismonoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of theC. churrovisstrain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.

Inhibition of spore germination offers an attractive and effective target for controlling fungal species involved in food spoilage. Mushroom alcohol (1-octen-3-ol) functions as a natural self-inhibitor of spore germination for many fungi and, therefore, provides a useful tool for probing the molecular events controlling the early stages of fungal growth. In Penicillium spp., the R and S enantiomers of 1-octen-3-ol delayed spore germination and sporulation in four species of Penicillium involved in soils of fruit and grains, but to different degrees. Because of its well-annotated genome, we used Penicillium chrysogenum to perform a comprehensive comparative transcriptomic analysis of cultures treated with the two enantiomers. Altogether, about 80% of the high-quality reads could be mapped to 11,396 genes in the reference genome. The top three active pathways were metabolic (978 transcripts), biosynthesis of secondary metabolites (420 transcripts), and microbial metabolism in diverse environments (318 transcripts). When compared to the control, treatment with (R)-(-)-1-octen-3-ol affected the transcription levels of 91 genes, while (S)-(+)-1-octen-3-ol affected only 41 genes. Most of the affected transcripts were annotated and predicted to be involved in transport, establishment of localization, and transmembrane transport. Alternative splicing and SNPs’ analyses indicated that, compared to the control, the R enantiomer had greater effects on the gene expression pattern of Penicillium chrysogenum than the S enantiomer. A qRT-PCR analysis of 28 randomly selected differentially expressed genes confirmed the transcriptome data. The transcriptomic data have been deposited in NCBI SRA under the accession number SRX1065226.

Many microorganisms produce volatile organic compounds (VOCs) with biological effects on plants. In this study, Arabidopsis seeds or 14-day-old vegetative plants were exposed to 0.5 μg/l of chemical standards of 26 VOCs previously identified from the biocontrol fungus Trichoderma. Seven compounds (1-decene, 2-heptylfuran, 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1- butanol, 2-heptanone, and 1-octen-3-ol) were further tested at the physiological concentration (10 ng/l) and 3-methyl-1-butanol, 1-decene, and 2-heptylfuran induced significant increases in fresh weight and total chlorophyll content. Plants exposed to 1-decene had the greatest increase in plant fresh shoot weight (38.9%) and chlorophyll content (67.8%). An RNA sequencing analysis was performed on plants treated with vapors of 1-decene. The expression of 123 genes was differentially affected, encompassing genes involved in cell wall modification, auxin induction, stress, and defense responses, with several major classes of stress-related genes showing down-regulation. To our knowledge, this is the first report of the effect of a plant growth promoting VOC on gene expression in Arabidopsis thaliana. As the role of fungal VOCs in biocontrol moves from correlative studies to more hypothesis driven approaches, our findings can guide both basic and applied studies in agricultural research.

Biofortification, a method for increasing micronutrient content of staple crops, is a promising strategy for combating major global health problems, such as iron and zinc deficiency. We examined the acceptability of recipes prepared using iron- and zinc-biofortified pearl millet (FeZnPM) (~80 ppm Fe, ~34 ppm Zn, varietal ICTP-8203), compared to conventional pearl millet (CPM) (~20 ppm Fe, ~19 ppm Zn) in preparation for an efficacy trial. Our objective was to examine the acceptability of FeZnPM compared to CPM among young children and mothers living in the urban slums of Mumbai. Standardized traditional feeding program recipes (n = 18) were prepared with either FeZnPM or CPM flour. The weight (g) of each food product was measured before and after consumption by children (n = 125) and the average grams consumed over a 3-day period were recorded. Mothers (n = 60) rated recipes using a 9-point hedonic scale. Mean intakes and hedonic scores of each food product were compared using t-tests across the two types of pearl millet. There were no statistically significant differences in consumption by children (FeZnPM: 25.27 ± 13.0 g; CPM: 21.72 ± 6.90 g) across the food products (P = 0.28). Overall mean hedonic scores for all recipes were between 7 to 9 points. CPM products were rated higher overall (8.22 ± 0.28) compared to FeZnPM products (7.95 ± 0.35) (P = 0.01). FeZnPM and CPM were similarly consumed and had high hedonic scores, demonstrating high acceptability in this population. These results support using these varieties of pearl millet in a proposed trial [http://Clinicaltrials.gov ID: NCT02233764; Clinical Trials Registry of India (CTRI), reference number REF/2014/10/007731, CTRI number CTRI/2015/11/006376] testing the efficacy of FeZnPM for improving iron status and growth.

In this paper, we demonstrate the ability of Arabidopsis thaliana to detect different mixtures of volatile organic compounds (VOCs) emitted by the common indoor fungus, Aspergillus versicolor, and demonstrate the potential usage of the plant as a bioindicator to monitor fungal VOCs in indoor air. We evaluated the volatile production of Aspergillus versicolor strains SRRC 108 (NRRL 3449) and SRRC 2559 (ATCC 32662) grown on nutrient rich fungal medium, and grown under conditions to mimic the substrate encountered in the built environment where fungi would typically grow indoors (moist wallboard and ceiling tiles). Using headspace solid phase microextraction/gas chromatography-mass spectrometry, we analyzed VOC profiles of the two strains. The most abundant compound produced by both strains on all three media was 1-octen-3-ol. Strain SRRC 2559 made several terpenes not detected from strain SRRC 108. Using a split-plate bioassay, we grew Arabidopsis thaliana in a shared atmosphere with VOCs from the two strains of Aspergillus versicolor grown on yeast extract sucrose medium. The VOCs emitted by SRRC 2559 had an adverse impact on seed germination and plant growth. Chemical standards of individual VOCs from the Aspergillus versicolor mixture (2-methyl-1-butanol, 3-methyl-1-butanol, 1-octen-3-ol, limonene, and β-farnesene), and β-caryophyllene were tested one by one in seed germination and vegetative plant growth assays. The most inhibitory compound to both seed germination and plant growth was 1-octen-3-ol. Our data suggest that Arabidopsis is a useful model for monitoring indoor air quality as it is sensitive to naturally emitted fungal volatile mixtures as well as to chemical standards of individual compounds, and it exhibits relatively quick concentration- and duration-dependent responses.

“Mushroom alcohol,” or 1-octen-3-ol, is a common fungal volatile organic compound (VOC) that has been studied for its flavor properties, its effects on fungal spore germination, mushroom development, and as a signaling agent for insects. Far less is known about its effects on plants. We exposed Arabidopsis thaliana seeds, under conditions conducive to germination, to high (10 and 100 mg/1) and low concentrations (1, 2, and 3 mg/1) of racemic, S, and R forms of 1-octen-3-ol for 3 days. In addition, 1-, 2-, 3-, and 4-week-old A. thaliana plants also were exposed to 1 mg/1 of the compounds for the same period of time. Seedling formation was retarded with all tested levels of exposure to 1-octen-3-ol for both enantiomers and the racemer, while 95% of unexposed control seeds germinated to seedling within 3 days. There was a dose-dependent response in the reduction of seedling formation between 1 mg/1 and 3 mg/1 of exposure. When exposed seeds were removed from the VOC, nearly all resumed germination. Young plants exposed to 1 mg/1 of the R and S enantiomers of 1-octen-3-ol exhibited a mild inhibition of growth and chlorophyll production at 2 and 3 weeks but not at 4 weeks.

Geosmin and C-8 hydrocarbons are among the major volatile organic compounds (VOC) responsible for the distinctive, musty odor of filamentous fungi. In this study, we developed a plant bioassay for testing the possible toxicity of these compounds, as well as four air freshener products sometimes used to mask their odor. Seeds and vegetative plants of Arabidopsis thaliana were exposed to 1 ppm of 14 different volatile treatments (both single compounds and mixtures) for 72 h and monitored for germination rate, seedling formation, vegetative plant vigor and chlorophyll concentration. All VOCs tested had some inhibitory effect on seed germination or seedling formation; 1-octen-3-one was the most active, giving almost complete inhibition of germination. Geosmin did not prevent germination (radicle protrusion) but seedling formation was arrested 90 %. Of solvents and fragrance products tested, only the scented oil product was as active as the C-8 biogenic compounds in inhibiting seed germination and seedling formation. Two-week-old plants exposed to 1 ppm of individual fungal VOCs for 72 h all exhibited some degree of stress symptoms including smaller leaf size and weight, discoloration, leaf curling, small necrotic lesions, and reduced chlorophyll concentration. Two-week-old vegetative plants exposed to solvents and air freshener products were generally smaller in size. The single most phytotoxic compound tested was 1-octen-3-one which almost completely inhibited seed germination and was lethal to vegetative phase plants. Formaldehyde at 1 ppm killed 2-week-old seedlings but had little effect on seed germination. In conclusion, the A. thaliana bioassay provides an inexpensive approach for testing the toxicity of gas phase molecules.

Members of the 14-3-3 family of proteins are conserved regulatory proteins that are widely found in eukaryotes and play crucial roles in diverse physiological processes, including responses to different stresses. Although genome-wide analysis of 14-3-3 proteins has been performed in a few plant species, it has not been performed in switchgrass. In this study, we identified 21 switchgrass 14-3-3 proteins (designated PvGF14a to PvGF14u) and examined genes for improved stress tolerance in this species. A phylogenetic tree was constructed to demonstrate that PvGF14 proteins can be divided into six groups, and that PvGF14 proteins belonging to each class exhibit similar gene structure. A phylogenetic analysis of PvGF14 proteins among switchgrass, Arabidopsis, and rice was conducted. Ten PvGF14 proteins were found to be orthologous to several abiotic stresses, and these were particularly responsive proteins in Arabidopsis and rice. Tissue-specific expression profiles showed that PvGF14a, PvGF14k, PvGF14l, and PvGF14m may play significant roles in the regulation of lignin metabolism, and that PvGF14r may participate in flower development. Taken together, these data suggest that PvGF14 proteins may be involved in various biosynthesis.

Previous studies have indicated that secondary plant metabolites may modulate glucose absorption in the small intestine. We have characterized a polyphenol-rich herbal extract and its potential intestinal metabolites by LC-MS(2) and investigated the inhibition of glucose transporters SGLT1 and GLUT2 using the well-characterized Caco-2 intestinal model. Differentiated Caco-2 monolayers were incubated with an extract of a mixture of herbs and spices. Glucose transport under sodium-dependent and sodium-free conditions was determined by radiochemical detection of D-[U-(14) C]-glucose. A 54% decrease in transport was observed compared to control. Using sodium-dependent and sodium-free conditions, we demonstrate that the inhibition of GLUT2 was greater than SGLT1. Glycosidase and esterase enzymatic hydrolysis was used to assess the impact of metabolism on the efficacy of inhibition. Glucose transport across the membrane was reduced by 70% compared to the control and was associated with significant increases in flavonoid aglycones, caffeic acid, and p-coumaric acid. These results suggest that intact and hydrolyzed polyphenols, likely to be found in the lumen after ingestion of the supplement, play an important role in the attenuation of glucose absorption and may have potentially beneficial antiglycemic effects in the body.

Arnon R, Annunziato R, Schiano T, Miloh T, Baisley M, Sogawa H, Contreras AG, Lee S, Kerkar N. Orthotopic liver transplantation for adults with Alagille syndrome. Clin Transplant 2011 DOI: 10.1111/j.1399-0012.2011.01574.x. © 2011 John Wiley & Sons A/S. Abstract: Introduction: Alagille syndrome (AGS) is an inherited multisystem disorder, and liver transplantation (LT) may be required in pediatric patients with AGS (P-AGS). There are limited data regarding the outcomes of LT in adults with AGS (A-AGS). Aim: To determine and compare the outcomes of LT in A-AGS vs. P-AGS as well as A-AGS vs. adults with biliary atresia (A-BA). Methods: Adults (>18 yr), with AGS and BA, and children (≤18 yr), with AGS who underwent isolated first LT between 10/1987 and 5/2008, were identified from the UNOS database. Results: Forty-four of 79 400 adults transplanted for AGS were compared with 407 P-AGS and 56 A-BA, respectively. A-AGS patients had a significantly higher rate of encephalopathy, lower serum albumin, and higher serum creatinine in comparison with P-AGS. One- and five-yr patient and graft survival in A-AGS who underwent LT were not significantly different in comparison with either P-AGS or A-BA (A-AGS patient survival: 95.5%, 90.9%, P-AGS: 88. 7%, 86.2%, A-BA: 89.3%, 87.5%; A-AGS graft survival: 84.1%, 79. 5%, P-AGS: 80.3%, 76%. 1%, A-BA: 82.1%, 78.6%, respectively). Conclusion: The outcome of first LT in A-AGS is excellent compared with the overall reported adult patient and graft survival. Although A-AGS were sicker than P-AGS at transplant, their outcomes were comparable with that of P-AGS.

Background Jasmonic acid (JA) and methyl jasmonate (MeJA) regulate plant development, resistance to stress, and insect attack by inducing specific gene expression. However, little is known about the mechanism of plant defense against herbivore attack at a protein level. Using a high-resolution 2-D gel, we identified 62 MeJA-responsive proteins and measured protein expression level changes. Results Among these 62 proteins, 43 proteins levels were increased while 11 proteins were decreased. We also found eight proteins uniquely expressed in response to MeJA treatment. Data are available via ProteomeXchange with identifier PXD001793. The proteins identified in this study have important biological functions including photosynthesis and energy related proteins (38.4%), protein folding, degradation and regulated proteins (15.0%), stress and defense regulated proteins (11.7%), and redox-responsive proteins (8.3%). The expression levels of four important genes were determined by qRT-PCR analysis. The expression levels of these proteins did not correlate well with their translation levels. To test the defense functions of the differentially expressed proteins, expression vectors of four protein coding genes were constructed to express in-fusion proteins in E. coli. The expressed proteins were used to feed Ostrinia furnacalis, the Asian corn borer (ACB). Our results demonstrated that the recombinant proteins of pathogenesis-related protein 1 (PR1) and thioredoxin M-type, chloroplastic precursor (TRXM) showed the significant inhibition on the development of larvae and pupae. Conclusions We found MeJA could not only induce plant defense mechanisms to insects, it also enhanced toxic protein production that potentially can be used for bio-control of ACB. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1363-1) contains supplementary material, which is available to authorized users.

All odorants are volatile organic compounds (VOCs), i.e., low molecular weight compounds that easily evaporate at normal temperatures and pressure. Fungal VOCs are relatively understudied compared to VOCs of bacterial, plant, or synthetic origin. Much of the research to date on fungal VOCs has focused on their food and flavor properties, their use as indirect indicators of fungal growth in agriculture, or their role as semiochemicals for insects. In addition, research into fungal volatiles has also taken place to monitor spoilage, for purposes of chemotaxonomy, for use in biofilters and for biodiesel, to detect plant and animal disease, for “mycofumigation,” and with respect to plant health. As methods for the analysis of gas phase molecules have improved, it has become apparent that fungal VOC are more chemically varied and more biologically active than has generally been realized. In particular, there is increasing data that show that fungal VOCs frequently mediate interactions between organisms within and across different ecological niches. The goal of this mini review is to orchestrate data on fungal VOCs obtained from disparate disciplines as well as to draw attention to the ecological importance of fungal VOCs in signaling between different species. Technologies and approaches that are common in one area of research are often unknown in others, and the study of fungal VOCs would benefit from more cross talk between subdisciplines.

Void nucleation in aluminum interconnects passivated with silicon nitride was studied using a high voltage scanning electron microscope. Extensive stress-induced voiding was observed in these interconnects independent of the passivation thickness. Some of the stress-induced and electromigration-induced voids formed away from the interconnect sidewall in contrast to results from other studies of void nucleation in passivated aluminum lines. Nuclear reaction analysis measured large amounts of hydrogen in the aluminum films passivated with silicon nitride. Transmission electron microscopy showed a high density of nanometer-sized bubbles in the aluminum. These bubbles, which are thought to have formed from hydrogen that evolved from the silicon nitride layer during processing, served as nucleation sites for stress- and electromigration-induced voids.

Fungal volatile organic compounds (VOCs) play important ecophysiological roles in mediating inter-kingdom signaling with arthropods but less is known about their interactions with plants. In this study, Arabidopsis thaliana was used as a model in order to test the physiological effects of 23 common vapor-phase fungal VOCs that included alcohols, aldehydes, ketones, and other chemical classes. After exposure to a shared atmosphere with the 23 individual VOCs for 72 hrs, seeds were assayed for rate of germination and seedling formation; vegetative plants were assayed for fresh weight and chlorophyll concentration. All but five of the VOCs tested (1-decene, 2-n-heptylfuran, nonanal, geosmin and -limonene) had a significant effect in inhibiting either germination, seedling formation or both. Seedling formation was entirely inhibited by exposure to 1-octen-3-one, 2-ethylhexanal, 3-methylbutanal, and butanal. As assayed by a combination of fresh weight and chlorophyll concentration, 2-ethylhexanal had a negative impact on two-week-old vegetative plants. Three other compounds (1-octen-3-ol, 2-ethylhexanal, and 2-heptylfuran) decreased fresh weight alone. Most of the VOCs tested did not change the fresh weight or chlorophyll concentration of vegetative plants. In summary, when tested as single compounds, fungal VOCs affected A. thaliana in positive, negative or neutral ways.

Cocoa is a dry, powdered, nonfat component product prepared from the seeds of the Theobroma cacao L. tree and is a common ingredient of many food products, particularly chocolate. Nutritionally, cocoa contains biologically active substances that may affect human health: flavonoids (epicatechin and oligomeric procyanidins), theobromine, and magnesium. Theobromine and epicatechin are absorbed efficiently in the small intestine, and the nature of their conjugates and metabolites are now known. Oligomeric procyanidins are poorly absorbed in the small intestine, but catabolites are very efficiently absorbed after microbial biotransformation in the colon. A significant number of studies, using in vitro and in vivo approaches, on the effects of cocoa and its constituent flavonoids have been conducted. Most human intervention studies have been performed on cocoa as an ingredient, whereas many in vitro studies have been performed on individual components. Approximately 70 human intervention studies have been carried out on cocoa and cocoa-containing products over the past 12 years, with a variety of endpoints. These studies indicate that the most robust biomarkers affected are endothelial function, blood pressure, and cholesterol level. Mechanistically, supporting evidence shows that epicatechin affects nitric oxide synthesis and breakdown (via inhibition of nicotinamide adenine di-nucleotide phosphate oxidase) and the substrate arginine (via inhibition of arginase), among other targets. Evidence further supports cocoa as a biologically active ingredient with potential benefits on biomarkers related to cardiovascular disease. However, the calorie and sugar content of chocolate and its contribution to the total diet should be taken into account in intervention studies.

Both fungi and bacteria emit many volatile organic compounds (VOCs) as mixtures of low molecular mass alcohols, aldehydes, esters, terpenoids, thiols, and other small molecules that easily volatilize. Most determination (separation and identification) of VOCs now relies on gas chromatography–mass spectrometry (GC-MS) but developments in “electronic nose” technology promise to revolutionize the field. Microbial VOC profiles are both complex and dynamic: the compounds produced and their abundance vary with the producing species, the age of the colony, water availability, the substrate, the temperature, and other environmental parameters. The single most commonly reported volatile from fungi is 1-octen-3-ol which is a breakdown product of linoleic acid. It functions as a hormone within many fungal species, serves as both an attractant and deterrent for certain species of arthropods, and exhibits toxicity at relatively low concentrations in model systems. Bacterial and fungal VOCs have been studied by scientists from a broad range of subdisciplines in both theoretical and applied contexts. VOCs are exploited for their food and flavor properties, their use as indirect indicators of microbial growth, their ability to stimulate plant growth, and their ability to attract insect pests. Because these compounds can diffuse a long way from their point of origin, they are excellent chemical signaling molecules (semiochemicals) in non-aqueous habitats and facilitate the ability of microbes to engage in “chemical conversations.” The physiological effects of bacterial and fungal VOCs in host–pathogen relationships and in mediating interspecific associations in natural ecosystem functioning is an emerging frontier for future research.

Void nucleation in nitride passivated aluminum interconnects was studied using High Voltage Scanning Electron Microscopy (HVSEM). Extensive stress-induced voiding was observed in the line regardless of passivation thickness. Some of the stress-induced and electromigration voids formed away from the interconnect sidewall, which is contrary to images presented in other studies of void nucleation. Transmission electron microscopy showed that hydrogen from the nitride layer forms bubbles in the aluminum. These bubbles act as pre-existing defects in the aluminum, reducing the barrier for void nucleation.

The electromigration behavior of pure Al lines passivated with oxides of different thicknesses and passivation deposition temperatures was studied. The initial hydrostatic stress states of the passivated Al lines were modeled with finite element modeling (FEM), and, when possible, measured with X-ray diffraction. Conventional wafer-level electromigration tests showed a clear passivation thickness effect, but no detectable effect of initial stress on electromigration lifetimes. Increasing the passivation thickness increased the electromigration lifetimes, which has been observed by other researchers. However, in a sample set where the Al lines were covered with a thin (0. 1µm) oxide layer, the lifetimes were much longer than expected. Differences in the damage morphology and the failure mechanism between the thin and thicker oxides accounted for this unexpected result.

Electromigration void behavior in Al-0.1%Sc and Al-0.5%Cu alloys was compared by conducting tests in a High Voltage Scanning Electron Microscope (HVSEM). Wafer level test results showed that the median time to failure for the Al-Cu lines was ten times longer than that of the Al-Sc lines. In-situ tests of the two alloys showed a similarity in void nucleation times but differences in void movement between the two sample sets; the interactions of voids with Al2Cu theta precipitates in Al-Cu lines were especially interesting. From our studies, it appeared that the addition of scandium to Al does not have the proposed benefits for electromigration resistance, as suggested by previous studies.

The average stress state and electromigration behavior of Al lines under 0.1μm oxide, 0.1μm oxide/1.0μm oxide, 0.1μm oxide/1.0μm nitride, or 0.1μm oxide/1.0μm polymer were analyzed. Thermal stresses calculated from finite element analysis and measured via x-rays were compared. A high voltage SEM was used to examine the stress samples for voiding and also to conduct in-situ electromigration testing on the various passivated lines. A reasonable correlation between the measured and calculated stresses was found for the unvoided samples, while results for the voided samples showed little correlation due to stress relaxation through voiding. Initial electromigration observations showed void behavior and failure modes varied under the different passivations.