Spore Cell Wall Research Articles

Fabrication and characterization of graphene/molecule/graphene vertical junctions with aryl alkane monolayers

We studied the effect of plasma treatment on the optical, structural and biological properties of Neurospora crassa (N. crassa) spores. An atmospheric-pressure plasma jet (APPJ) was used to generate reactive oxygen and nitrogen species in aqueous solution. The APPJ treatment of N. crassa spores in water significantly reduced the viability of spores. The reduction in the spore viability can be attributed to the reactive species from the plasma itself and those derived from the reaction of plasma radicals with aqueous solution. These structural modifications were contingent on the medium in which N. crassa spores were suspended; plasma treatment of N. crassa spores in PBS did not significantly affect the viability of spores as compared with N. crassa spores in water. Scanning electron microscopy images and circular dichroism spectra indicated that the spore cell wall was damaged by plasma treatment. The optical absorption spectrum of untreated N. crassa spores exhibited two resonance absorption bands at approximately λ1 ≈ 260 nm and λ2 ≈ 472 nm, originating from deoxyribonucleic acid (DNA) and β-carotene. The Raman spectrum of untreated N. crassa spores exhibited three main peaks at 1519, 1157 and 1006 cm −1, attributed to β-carotene inside the cell wall. The Raman spectra showed that the APPJ treatment of N. crassa spores in water caused degradation of β-carotene, affecting the viability of spores.

Activity of chitosan-lysozyme nanoparticles on the growth, membrane integrity, and β-1,3-glucanase production by Aspergillus parasiticus.

Synthesis of nanocomposites from antimicrobial biopolymers such as chitosan (CS) and lysozyme (LZ) is an important and promising area in bionanotechnology. Chitosan-lysozyme (CS-LZ) nanoparticles (NPs) were prepared by the nanoprecipitation method, using commercial chitosan of 153kDa. TEM and dynamic light scattering (DLS) analysis were carried out to evaluate the morphology, size, dispersion, and Z potential. Association efficiency of lysozyme was determined using Coomassie blue assay. The antifungal activity of NPs against Aspergillus parasiticus was evaluated through cell viability (XTT), germination and morphometry of spores, and reducing sugars production; the effects on membrane integrity and cell wall were also analyzed. NPs' size were found in the range of 13.4 and 11.8nm for CS-LZ and CS NPs, respectively, and high Z potential value was observed in both NPs. Also, high association of lysozyme was presented in the CS matrix. With respect to the biological responses, CS-LZ NPs reduced the viability of A. parasiticus and a strong inhibitory effect on the germination of spores (100% of inhibition) was observed at 24h in in vitro assays. CS-LZ and CS NPs affected the membrane integrity and the cell wall of spores of fungi with respect to control, which is consistent with the low amount of reducing sugars detected. CS-LZ NPs prepared by nanoprecipitation promise to be a viable and safe alternative for use in biological systems, with a possible low or null impact to humans and biota. However, the potential benefits and the environmental and health implications of NPs need to be globally discussed due to its possible negative effects.

Short-Chain Chitin Oligomers: Promoters of Plant Growth.

Chitin is the second most abundant biopolymer in nature after cellulose, and it forms an integral part of insect exoskeletons, crustacean shells, krill and the cell walls of fungal spores, where it is present as a high-molecular-weight molecule. In this study, we showed that a chitin oligosaccharide of lower molecular weight (tetramer) induced genes in Arabidopsis that are principally related to vegetative growth, development and carbon and nitrogen metabolism. Based on plant responses to this chitin tetramer, a low-molecular-weight chitin mix (CHL) enriched to 92% with dimers (2mer), trimers (3mer) and tetramers (4mer) was produced for potential use in biotechnological processes. Compared with untreated plants, CHL-treated plants had increased in vitro fresh weight (10%), radicle length (25%) and total carbon and nitrogen content (6% and 8%, respectively). Our data show that low-molecular-weight forms of chitin might play a role in nature as bio-stimulators of plant growth, and they are also a known direct source of carbon and nitrogen for soil biomass. The biochemical properties of the CHL mix might make it useful as a non-contaminating bio-stimulant of plant growth and a soil restorer for greenhouses and fields.

Beyond animals and plants: dynamic maternal effects in the fungus Neurospora crassa.

Maternal effects are widely documented in animals and plants, but not in fungi or other eukaryotes. A principal cause of maternal effects is asymmetrical parental investment in a zygote, creating greater maternal vs. paternal influence on offspring phenotypes. Asymmetrical investments are not limited to animals and plants, but are also prevalent in fungi and groups including apicomplexans, dinoflagellates and red algae. Evidence suggesting maternal effects among fungi is sparse and anecdotal. In an experiment designed to test for maternal effects across sexual reproduction in the model fungus Neurospora crassa, we measured offspring phenotypes from crosses of all possible pairs of 22 individuals. Crosses encompassed reciprocals of 11 mating-type 'A' and 11 mating-type 'a' wild strains. After controlling for the genetic and geographic distances between strains in any individual cross, we found strong evidence for maternal control of perithecia (sporocarp) production, as well as maternal effects on spore numbers and spore germination. However, both parents exert equal influence on the percentage of spores that are pigmented and size of pigmented spores. We propose a model linking the stage-specific presence or absence of maternal effects to cellular developmental processes: effects appear to be mediated primarily through the maternal cytoplasm, and, after spore cell walls form, maternal influence on spore development is limited. Maternal effects in fungi, thus far largely ignored, are likely to shape species' evolution and ecologies. Moreover, the association of anisogamy and maternal effects in a fungus suggests maternal effects may also influence the biology of other anisogamous eukaryotes.

Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination in Streptomyces coelicolor.

This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall.

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.

Carbon Sequestration by Glomeral Fungi in Soil Is Influenced by Phosphorus and Nitrogen Fertilization

Glomalin is the most abundant gylcoprotein in soil which produces by glomeral fungi in symbiosis with plant roots. It improves soil physical, chemical and biological properties. Assimilated plant C which is allocated to the mycorrhizal fungus, appears as a recalcitrant glycoprotein (glomalin) in cell walls of hyphae and spores. Considering global warming due to increasing greenhouse gases, this phenomenon could be important in carbon sequestration and reducing CO 2 in atmosphere. Chemical fertilizers could affect symbiotic relations of these fungi, which in turn affect glomalin production. In a pot culture experiment, the sterile soil was treated with 0, 100, 200 mg N kg -1 soil as urea or 0, 20, 40 mg P kg -1 soil as triple superphosphate, in two separate factorial experiment based on completely randomized design with three replications. Corn plant ( Zea mays L.) was inoculated with Rhizophagus clarus (formerly, Glomus clarum ) or Rhizophagus intraradices (formerly, Glomus intraradices ) in each set of experiment. Easley extractable glomalin (EEG) and total glomalin (TG) in soil were determined by Bradford method at the end of experiment. Root colonization by both fungi increased EEG and TG compared to the non-mycorrhizal control ( p <0.05). Nitrogen levels of 100, 200 increased EEG by 75 and 112% and TG by 59 and 76%, respectively, compared to the no nitrogen treatment. P levels of 20, 40 caused 27% increase and 6% decrease in EEG, and 24% increase and 13% decrease in TG, respectively, compared to the zero addition of phosphorus. Regarding glomalin production in this condition, R. clarus was more efficient than R. intraradices. Application of 20 mg P kg -1 increased root colonization, dry weights of shoot and root, chlorophyll index, leaf area, amount of shoot and root nitrogen and potassium compared to the 40 mgP. kg -1 and control. Thus, application of 100 mg N kg -1 increased root colonization, dry weights of shoot and root, chlorophyll index, leaf area, amount of shoot and root phosphorus and potassium compared to the 200 mg N kg -1 and control.

Systems approaches to predict the functions of glycoside hydrolases during the life cycle of Aspergillus niger using developmental mutants ∆brlA and ∆flbA.

BackgroundThe filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis.ResultsIn this study, we used developmental mutants (ΔflbA and ΔbrlA) which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced.ConclusionThe combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall modification, which contributes to understanding cell wall remodeling mechanisms during development.

Alternaria alternata transcription factor CmrA controls melanization and spore development.

Melanin is a black pigment widely distributed across the kingdoms, from bacterial to human. The filamentous fungus Alternaria alternata is a typical 'black fungus', which produces melanin in its hyphal and especially its asexual spore cell walls. Its biosynthesis follows the dihydroxynaphthalene (DHN) pathway with 1,8-DHN as an intermediate. Two genes, encoding a polyketide synthase (pksA) and a 1,3,8-trihydroxynaphthalene (THN) reductase (brm2), along with a putative transcription factor, CmrA, comprise a small gene cluster. Here we show that CmrA controls the expression of pksA and brm2, but that it also controls the expression of a scytalone dehydratase encoding gene (brm1) located elsewhere in the genome. The regulatory function of CmrA was shown in a reporter assay system. Al. alternata CmrA was expressed in the filamentous fungus Aspergillus nidulans where it was able to induce the expression of a reporter construct under the control of the putative pksA promoter. This suggests direct binding of CmrA to the promoter of pksA in the heterologous system. Likewise, silencing of cmrA in Al. alternata led to white colonies due to the lack of melanin. In addition, hyphal diameter and spore morphology were changed in the mutant and the number of spores reduced. Silencing of brm2 and inhibition of melanin biosynthesis by tricyclazole largely phenocopied the effects of cmrA silencing, suggesting a novel regulatory function of melanin in morphogenetic pathways.

Marine Streptomyces cyaneus Strain Alex-SK121 Mediated Eco-friendly Synthesis of Silver Nanoparticles Using Gamma Radiation

Aim:&nbsp;The present study aimed to develop cost-effective, eco-friendly marine&nbsp;Streptomyces cyaneus&nbsp;strain Alex-SK121 mediated synthesis of silver nanoparticles (AgNPs) with antimicrobial, antitumor and antioxidant activities.Methodology:&nbsp;Aqueous 1mM silver nitrate (AgNO3) solution was treated with cell-free supernatant (CFS) of a novel&nbsp;Streptomyces cyaneus&nbsp;strain Alex-SK121 isolated from marine sediment samples. The prepared solution was irradiated with different doses of gamma rays ranged from 0.5 to 30.0kGy. Initial characterization of the synthesized AgNPs was performed by visual observation of color change in the prepared solution followed by analysis of UV-Visible Spectrophotometer (UV-Vis.), Fourier Transform Infrared Spectrometer (FT-IR), Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). Evaluation of antimicrobial activity of the synthesized AgNPs against some pathogenic microorganisms was carried out. Antitumor activity of AgNPs was carried out against some human cancer cell lines using the method of Sulphorodamine B (SRB) assay, antioxidant activity of AgNPs was also studied using DPPH scavenging assay.Results:&nbsp;In the present study, the cell-free supernatant of&nbsp;Streptomyces cyaneus&nbsp;strain Alex-SK121 isolated from sediment samples collected from Sidi Kerir region, Alexandria governorate, Egypt was found to reduce Ag+&nbsp;ions to AgNPs. Identification of the producer strain was performed according to spore morphology and cell wall chemo-type, which suggested that this strain is Streptomyces. Further cultural, physiological characteristics and analysis of the nucleotide sequence of 16S rRNA gene indicated that this strain is identical to&nbsp;Streptomyces cyaneus&nbsp;and then designated&nbsp;Streptomyces cyaneus&nbsp;strain Alex-SK121. To maximize the production of AgNPs, the tested supernatant was irradiated with different doses of gamma rays and it was found that, 15 kGy is the best applied dose induces AgNPs synthesis. The synthesized AgNPs showed the characteristic absorption spectra in UV–Vis. at 425 nm. The microbiologically synthesized AgNPs showed significant antimicrobial activity towards some pathogenic microorganisms with inhibition zone ranged from 13 up to 20 mm. Also AgNPs exhibited antitumor activity against human breast carcinoma cells and human liver carcinoma cells with IC50&nbsp;9.63 and 33.75 µg/ml respectively in addition to 96% antioxidant activity.Conclusion:&nbsp;Gamma irradiation which induced AgNPs synthesis by cell-free supernatant of marine actinomycetes&nbsp;Streptomyces cyaneus&nbsp;strain Alex-SK121 with different applications is a simple, clean, economic and environmental friendly approach.

Differentiation of Nosema apis and Nosema ceranae spores under Scanning Electron Microscopy (SEM)

SummarySpores of the microsporidian gut pathogens of honey bees Nosema apis and Nosema ceranae, the causative agents of nosemosis, are difficult to distinguish using the light microscope. This study aimed to find methods which could help to identify N. apis and N. ceranae spores, and to define morphometric parameters in order to distinguish the differences between the spores of the two species. Our observation of spores under Scanning Electron Microscopy (SEM) revealed that N apis and N. ceranae spores differ in their surface structure. Generally, N. ceranae spores seem to be more sculptured with deeper ornamentation than those of N. apis. Therefore, ornamentation of the spore cell wall with special reference to the width of their exospore furrows and sculpture index allows unequivocal identification of N. apis and N. ceranae species. Whole glands, organs, or various surfaces can be examined under SEM for the presence of spores, and consequently possible reservoirs or niches for storage of Nosema spores can be detected. We observed the results of Nosema spp. infestation on bees' intestines under SEM. Our study revealed that even medium Nosema spp. infection causes the midgut to be completely covered with spores. This sporemade layer can be the cause of bee malnutrition and higher mortality of foragers.

English

In the past few decades, nanoparticles have emerged as a field in biomedical research. Four isolated Aspergillus species were tested for extracellular synthesis of silver nanoparticles using their cell free filtrate (CFF). Silver nanoparticles of the most potent producer, Aspergillus terreus, were further characterized. Transmission electron microscope (TEM) and atomic force microscope (AFM) revealed their spherical shape, homog eneity and size range between 20 and 140 nm. X-ray diffraction (XRD) showed the crystalline nature of the biogenic silver nanoparticles. Fourier transform infra-red (FTIR) spectroscopic analysis indicated that the coordination behaviors between amino groups of the secreted fungal proteins and other functional groups present in the CFF may be liable for the reduction of silver ions to form stabilized protein-capped silver nanoparticles. They were stable in aqueous solution for four months of storage at room temperature under dark conditions. The biogenic silver nanoparticles showed remarkable antifungal activity against the human pathogenic fungus A. fumigatus. The spore cell wall, plasma membrane and the inner constituents were damaged as shown by TEM. Furthermore, comet assay proved high breakage of DNA. &nbsp; Key words: Silver nanoparticles, biosynthesis, fungi, antifungal, comet assay.

The Possible Role of SCO3388, a tmrB-like Gene of Streptomyces coelicolor, in Germination and Stress Survival of Spores

The SCO3388 gene from Streptomyces coelicolor is homologous to tmrB, the tunicamycin resistance gene of Bacillus subtilis. The SCO3388-inactivation strain (SY-tbl-1) was generated by replacing SCO3388 with thiostrepton resistance gene. Spores of S. coelicolor derivatives were prepared on mannitol-soy flour (MS) agar on which SY-tbl-1 displayed no significant defect in growth and development. When plated on R4 agar, spores of SYtbl-1 displayed retardation in growth and sporulation, whereas its mycelium gave rise to normal growth. Thus, SCO3388 is suggested to be involved in the dormant spore germination. Expression of SCO3388 under the ermE1 promoter restored but only partially the ability to sporulate in SY-tbl-1. Neither SY-tbl-1 nor SY-tbl-1/ermE1p-SCO3388 showed a difference in tunicamycin resistance to the wild type whereas, interestingly, the introduction of ermE1p-SCO3388 dramatically enhanced spore survival to heat and detergent treatments, suggesting that SCO3388 might play a role in the maintenance of spore cell wall integrity.

Chitinases CtcB and CfcI modify the cell wall in sporulating aerial mycelium of Aspergillus niger

Sporulation is an essential part of the life cycle of the industrially important filamentous fungus Aspergillus niger. The formation of conidiophores, spore-bearing structures, requires remodelling of the fungal cell wall, as demonstrated by the differences in carbohydrate composition of cell walls of vegetative mycelium and spores. Glycoside hydrolases that are involved in this process have so far remained unidentified. Using transcriptome analysis, we have identified genes encoding putative cell-wall-modifying proteins with enhanced expression in sporulating aerial mycelium compared to vegetative mycelium. Among the most strongly induced genes were those encoding a protein consisting of a putative chitin binding module (CBM14) and the chitinolytic enzymes NagA, CfcI and CtcB. Reporter studies showed that the N-acetyl-β-hexosaminidase gene nagA was expressed both in vegetative hyphae and in aerial structures (aerial hyphae, conidiophores and conidia) upon starvation. In contrast, promoter activities of the chitinase genes ctcB and cfcI were specifically localized in the conidiophores and conidia. CtcB is an endo-chitinase and CfcI releases monomers from chitin oligosaccharides: together these enzymes have the potential to degrade chitin of the fungal cell wall. Inactivation of both the cfcI and ctcB genes affected neither radial growth rate, nor formation and germination of spores. The amount of chitin in the spore walls of a ΔcfcIΔctcB double deletion strain, however, was significantly increased compared with the wild-type, thus indicating that CfcI and CtcB indeed modify the A. niger cell walls during sporulation. These novel insights in the sporulation process in aspergilli are of strong scientific relevance, and also may aid industrial strain engineering.

Complement Component 3C3 and C3a Receptor Are Required in Chitin-Dependent Allergic Sensitization to Aspergillus fumigatus but Dispensable in Chitin-Induced Innate Allergic Inflammation

ABSTRACTLevels of the anaphylatoxin C3a are increased in patients with asthma compared with those in nonasthmatics and increase further still during asthma exacerbations. However, the role of C3a during sensitization to allergen is poorly understood. Sensitization to fungal allergens, such as Aspergillus fumigatus, is a strong risk factor for the development of asthma. Exposure to chitin, a structural polysaccharide of the fungal cell wall, induces innate allergic inflammation and may promote sensitization to fungal allergens. Here, we found that coincubation of chitin with serum or intratracheal administration of chitin in mice resulted in the generation of C3a. We established a model of chitin-dependent sensitization to soluble Aspergillus antigens to test the contribution of complement to these events. C3−/− and C3aR−/− mice were protected from chitin-dependent sensitization to Aspergillus and had reduced lung eosinophilia and type 2 cytokines and serum IgE. In contrast, complement-deficient mice were not protected against chitin-induced innate allergic inflammation. In sensitized mice, plasmacytoid dendritic cells from complement-deficient animals acquired a tolerogenic profile associated with enhanced regulatory T cell responses and suppressed Th2 and Th17 responses specific for Aspergillus. Thus, chitin induces the generation of C3a in the lung, and chitin-dependent allergic sensitization to Aspergillus requires C3aR signaling, which suppresses regulatory dendritic cells and T cells and induces allergy-promoting T cells.

Inhibition of non-toxigenic Aspergillus niger FS10 isolated from Chinese fermented soybean on growth and aflatoxin B1 production by Aspergillus flavus

A non-toxigenic strain FS10 was isolated from Chinese fermented soybean to control AFB1 contamination and conformed as Aspergillus niger by 18S rDNA. To elucidate the biocontrol mechanism, the ability of culture filtrate of strain FS10 against growth and AFB1 production by toxigenic Aspergillus flavus was evaluated in vitro. Meanwhile, the influence of filtrate on spore morphology of A. flavus was analyzed by optical microscope and atomic force microscope (AFM). Results demonstrated that the culture filtrate of FS10 significantly suppressed A. flavus growth, sporulation and AFB1 biosynthesis. After 9 days, the inhibition of mycelia growth, sporulation and AFB1 production reached to 37.7%, 100% and 94.5%, respectively. In addition, optical microscopy and AFM showed that the culture filtrate serious damaged spore morphology, accompanying with spore analysis and cell wall collapse, resulted in inhibition of spore germination. The degradation activity of FS10 culture filtrate on AFB1 was also estimated. When AFB1 was incubated with the culture filtrate, 85.8% of AFB1 could be degraded after 5 days. It is concluded that the culture filtrate of strain FS10 has pleiotropic effects on control AFB1 contamination. As a safe, effective and economic biological agent, the filtrate could be applied in fermented food to prevent toxigenic Aspergillus spp. and AFB1 contamination.

MALDI‐based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI-TOF MS-based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer-based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5 × 10(9) spores per ml. The best peptide/protein profiles (in terms of signal-to-noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves.

Hydrogen and Oxygen Isotope Ratios in Body Water and Hair: Modeling Isotope Dynamics in Nonhuman Primates

The stable isotopic composition of drinking water, diet, and atmospheric oxygen influence the isotopic composition of body water ((2)H/(1)H, (18)O/(16)O expressed as δ(2) H and δ(18)O). In turn, body water influences the isotopic composition of organic matter in tissues, such as hair and teeth, which are often used to reconstruct historical dietary and movement patterns of animals and humans. Here, we used a nonhuman primate system (Macaca fascicularis) to test the robustness of two different mechanistic stable isotope models: a model to predict the δ(2)H and δ(18)O values of body water and a second model to predict the δ(2)H and δ(18)O values of hair. In contrast to previous human-based studies, use of nonhuman primates fed controlled diets allowed us to further constrain model parameter values and evaluate model predictions. Both models reliably predicted the δ(2)H and δ(18)O values of body water and of hair. Moreover, the isotope data allowed us to better quantify values for two critical variables in the models: the δ(2)H and δ(18)O values of gut water and the (18)O isotope fractionation associated with a carbonyl oxygen-water interaction in the gut (α(ow)). Our modeling efforts indicated that better predictions for body water and hair isotope values were achieved by making the isotopic composition of gut water approached that of body water. Additionally, the value of α(ow) was 1.0164, in close agreement with the only other previously measured observation (microbial spore cell walls), suggesting robustness of this fractionation factor across different biological systems.

A novel water-soluble β-d-glucan isolated from the spores of Ganoderma lucidum

Ganoderma lucidum is an edible and medicinal mushroom used widely in East Asia. In recent years, its spores have been used as a supplement in combination with other forms of antitumor therapies. The cell wall of Ganoderma lucidum spores contains a high amount of polysaccharides. In this study, a neutral polysaccharide, GLSA50-1B, was isolated from sporoderm-broken spores of Ganoderma lucidum, by hot-water extraction, graded ethanol precipitation, anion-exchange chromatography, and gel permeation chromatography. Using sugar compositional analysis, methylation analysis, partial acid hydrolysis, acetolysis, and NMR and ESI-MS spectroscopy, GLSA50-1B was elucidated to be a novel β-d-glucan featured by a 1,6-linked β-d-Glcp backbone with different length of branches consisting of terminal and 1,4-linked Glcp residues, attached to O-4 of alternative Glc residues in the backbone.

Predominant localization of the major Alternaria allergen Alt a 1 in the cell wall of airborne spores

Predominant localization of the major Alternaria allergen Alt a 1 in the cell wall of airborne spores