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In the last three decades, the scope of solid-state NMR has expanded to exploring complex biomolecules, from large protein assemblies to intact cells at atomic-level resolution. This diversity in macromolecules frequently features highly flexible components whose insoluble environment precludes the use of solution NMR to study their structure and interactions. While High-resolution Magic-Angle Spinning (HR-MAS) probes offer the capacity for gradient-based 1H-detected spectroscopy in solids, such probes are not commonly used for routine MAS NMR experiments. As a result, most exploration of the flexible regime entails either 13C-detected experiments, the use of partially perdeuterated systems, or ultra-fast MAS. Here we explore proton-detected pulse schemes probing through-bond 13C- 13C networks to study mobile protein sidechains as well as polysaccharides in a broadband manner. We demonstrate the use of such schemes to study a mixture of microtubule-associated protein (MAP) tau and human microtubules (MTs), and the cell wall of the fungus Schizophyllum commune using 2D and 3D spectroscopy, to show its viability for obtaining unambiguous correlations using standard fast-spinning MAS probes at high and ultra-high magnetic fields.

Carbon storage and cycling in boreal forests—the largest terrestrial carbon store—is moderated by complex interactions between trees and soil microorganisms. However, existing methods limit our ability to predict how changes in environmental conditions will alter these associations and the essential ecosystem services they provide. To address this, we developed a metatranscriptomic approach to analyze the impact of nutrient enrichment on Norway spruce fine roots and the community structure, function, and tree–microbe coordination of over 350 root-associated fungal species. In response to altered nutrient status, host trees redefined their relationship with the fungal community by reducing sugar efflux carriers and enhancing defense processes. This resulted in a profound restructuring of the fungal community and a collapse in functional coordination between the tree and the dominant Basidiomycete species, and an increase in functional coordination with versatile Ascomycete species. As such, there was a functional shift in community dominance from Basidiomycetes species, with important roles in enzymatically cycling recalcitrant carbon, to Ascomycete species that have melanized cell walls that are highly resistant to degradation. These changes were accompanied by prominent shifts in transcriptional coordination between over 60 predicted fungal effectors, with more than 5,000 Norway spruce transcripts, providing mechanistic insight into the complex molecular dialogue coordinating host trees and their fungal partners. The host–microbe dynamics captured by this study functionally inform how these complex and sensitive biological relationships may mediate the carbon storage potential of boreal soils under changing nutrient conditions.

Here, cell wall of a zygomycete fungus, Rhizopus delemar, grown on bread waste was wet spun into monofilaments. Using the whole cell wall material omits the common chitosan isolation and purification steps and leads to higher material utilization. The fungal cell wall contained 36.9% and 19.7% chitosan and chitin, respectively. Solid state NMR of the fungal cell wall material confirmed the presence of chitosan, chitin, and other carbohydrates. Hydrogels were prepared by ultrafine grinding of the cell wall, followed by addition of lactic acid to protonate the amino groups of chitosan, and subsequently wet spun into monofilaments. The monofilament inhibited the growth of Bacillus megaterium (Gram+ bacterium) and Escherichia coli (Gram- bacterium) significantly (92.2% and 99.7% respectively). Cytotoxicity was evaluated using an in vitro assay with human dermal fibroblasts, indicating no toxic inducement from exposure of the monofilaments. The antimicrobial and biocompatible fungal monofilaments, open new avenues for sustainable biomedical textiles from abundant food waste., QC 20230111

The fungi are an enormously successful eukaryotic lineage that has colonized every aerobic habitat on Earth. This spectacular expansion is reflected in the dynamism and diversity of the fungal cell wall, a matrix of polysaccharides and glycoproteins pivotal to fungal life history strategies and a major target in the development of antifungal compounds. Cell wall polysaccharides are typically synthesized by Leloir glycosyltransferases, enzymes that are notoriously difficult to characterize, but their nucleotide-sugar substrates are well known and provide the opportunity to inspect the monosaccharides available for incorporation into cell wall polysaccharides and glycoproteins. In this work, we have used phylogenomic analyses of the enzymatic pathways that synthesize and interconvert nucleotide-sugars to predict potential cell wall monosaccharide composition across 491 fungal taxa. The results show a complex evolutionary history of these cell wall enzyme pathways and, by association, of the fungal cell wall. In particular, we see a significant reduction in monosaccharide diversity during fungal evolution, most notably in the colonization of terrestrial habitats. However, monosaccharide distribution is also shown to be varied across later-diverging fungal lineages. IMPORTANCE This study provides new insights into the complex evolutionary history of the fungal cell wall. We analyzed fungal enzymes that convert sugars acquired from the environment into the diverse sugars that make up the fundamental building blocks of the cell wall. Species-specific profiles of these nucleotide-sugar interconverting (NSI) enzymes for 491 fungi demonstrated multiple losses and gains of NSI proteins, revealing the rich diversity of cell wall architecture across the kingdom. Pragmatically, because cell walls are essential to fungi, our observations of variation in sugar diversity have important implications for the development of antifungal compounds that target the sugar profiles of specif, QC 20210607

To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches - the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis, have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto, primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish "natto." Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto, as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied wi, QC 20200624

Unsold bread makes up a signification fraction of waste occurring in Swedish supermarkets. This thesis seeks to address the problem of food waste, by cultivating filamentous fungi on bread waste and producing chitinous films and nonwovens from them. Rhizopus delemar was cultivated on bread waste in liquid-state fermentation in order to obtain mycelia biomass. The biomass was processed by alkali or protease treatments to disrupt the fungal cells and remove proteins and fats. Afterwards it was subjected to a bleaching treatment to remove lignin fractions of bread residues. The treated biomass was then subjected to a grinding treatment for a homogeneous dispersion of mycelial fibers, where the dispersion was confirmed by microscopic images. The chemically and mechanically processed biomass was used for the preparation of films and nonwoven composites by employing a wet-laid papermaking process. The films exhibited plastic-like features, due to their brittleness and their smooth upper surface. Films and nonwoven composites were characterized on their tensile properties, surface water contact angle and their surface morphology by scanning electron microscopy. Treating fungal biomass by alkali and then bleaching resulted in films with atensile modulus of 3.38 GPa and an ultimate tensile strength of 71.50 MPa. These are the highest reported tensile properties for mycelia derived films to date. Water contact angle measurements confirmed a hydrophobic quality of mycelial films. Scanning electron microscopy showed a very dense and even surface without an obvious fibrous morphology. Fungal biomass and viscose fibers together form a rigid nonwoven composite, in which fungal biomass takes over the role of a natural eco-friendly binding matrix. Flexural rigidity measurements were out of bounds and need to be confirmed by future studies. Additionally, a second strain of fungi, Fusarium venenatum, was cultivated on bread particles in water suspension in order to determine optimum g

Unsold bread makes up a signification fraction of waste occurring in Swedish supermarkets. This thesis seeks to address the problem of food waste, by cultivating filamentous fungi on bread waste and producing chitinous films and nonwovens from them. Rhizopus delemar was cultivated on bread waste in liquid-state fermentation in order to obtain mycelia biomass. The biomass was processed by alkali or protease treatments to disrupt the fungal cells and remove proteins and fats. Afterwards it was subjected to a bleaching treatment to remove lignin fractions of bread residues. The treated biomass was then subjected to a grinding treatment for a homogeneous dispersion of mycelial fibers, where the dispersion was confirmed by microscopic images. The chemically and mechanically processed biomass was used for the preparation of films and nonwoven composites by employing a wet-laid papermaking process. The films exhibited plastic-like features, due to their brittleness and their smooth upper surface. Films and nonwoven composites were characterized on their tensile properties, surface water contact angle and their surface morphology by scanning electron microscopy. Treating fungal biomass by alkali and then bleaching resulted in films with atensile modulus of 3.38 GPa and an ultimate tensile strength of 71.50 MPa. These are the highest reported tensile properties for mycelia derived films to date. Water contact angle measurements confirmed a hydrophobic quality of mycelial films. Scanning electron microscopy showed a very dense and even surface without an obvious fibrous morphology. Fungal biomass and viscose fibers together form a rigid nonwoven composite, in which fungal biomass takes over the role of a natural eco-friendly binding matrix. Flexural rigidity measurements were out of bounds and need to be confirmed by future studies. Additionally, a second strain of fungi, Fusarium venenatum, was cultivated on bread particles in water suspension in order to determine optimum g

Fungi possess extraordinary strength in attachment to biotic and abiotic surfaces. This review focuses on adhesion mechanisms of yeast and filamentous fungi and the proposed combination of the adhesive forces of both organisms in an immobilization system called yeast biocapsules, whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum. The natural adherent properties of each organism, one multicellular and another unicellular, allow yeast to be fixated securely on the filamentous fungi and complete alcoholic fermentation. Following alcoholic fermentation, the hyphae become an inert support for yeast cells while maintaining shape and integrity. Biocapsules have been used successfully in both wine and bioethanol production. Investigation of the potential genes involved in fungal-yeast fusion suggests that natural hydrophobic interactions of both organisms play a major role. Analysis of the possible mechanisms involved in fungus and yeast adhesion, future perspectives on improving yeast immobilization, and proposed applications of the biocapsules are explored.

Fungi possess extraordinary strength in attachment to biotic and abiotic surfaces. This review focuses on adhesion mechanisms of yeast and filamentous fungi and the proposed combination of the adhesive forces of both organisms in an immobilization system called yeast biocapsules, whereby Saccharomyces cerevisiae cells are attached to the hyphae of Penicillium chrysogenum. The natural adherent properties of each organism, one multicellular and another unicellular, allow yeast to be fixated securely on the filamentous fungi and complete alcoholic fermentation. Following alcoholic fermentation, the hyphae become an inert support for yeast cells while maintaining shape and integrity. Biocapsules have been used successfully in both wine and bioethanol production. Investigation of the potential genes involved in fungal-yeast fusion suggests that natural hydrophobic interactions of both organisms play a major role. Analysis of the possible mechanisms involved in fungus and yeast adhesion, future perspectives on improving yeast immobilization, and proposed applications of the biocapsules are explored.

Fungal pathogens are the major cause of decay during postharvest handling and storage of fresh produce. Among the plethora of different fungi causing decay, necrotrophic fungi represent the main threat to major fruit crops, such as citrus, pome, or stone fruits. Classically, plant necrotrophic fungi have been considered unsophisticated pathogens that kill host cells with the use of toxins and/or cell wall-degrading enzymes (CWDE). However, studies conducted in recent years, mainly with Botrytis cinerea Pers. and Sclerotinia sclerotiorum (Lib.) de Bary are changing this simplistic perception about necrotrophic fungi. Consequently, there is an increased interest in elucidating the pathogenicity mechanisms deployed by these fungi. A recent review covered the molecular aspects of the interactions between postharvest pathogenic fungi and fruits, attending to both the pathogen and the host (Tian et al., 2016). This chapter is more focused on the current knowledge of the molecular mechanisms deployed by necrotrophic postharvest fungi.

[Background] In our previous study the synergism of four combinations of Zuccagnia punctata (ZpE) and Larrea nitida (LnE) exudates with the reliable statistical-based MixLow method was assessed, and the markers of the most anti-C. albicans synergistic ZpE-LnE bi-herbal combination were quantified according to European Medicines Agency (EMA)., [Purpose] To study the mechanisms of action as well as the cytotoxic properties of the ZpE-LnE most synergistic combination found in the previous work., [Materials and methods] Minimum Fungicidal Concentration (MFC) and rate of killing of ZpE-LnE were assessed with the microbroth dilution and the time-kill assays respectively. Morphological alterations were observed with both confocal and fluorescence microscopy on the yeast Schizosaccharomyces pombe. The ergosterol exogenous assay, the quantification of ergosterol, the sorbitol as well as glucan synthase (GS) and chitin synthase (ChS) assays were used to detect the effects on the fungal membrane and cell wall respectively. The capacity of ZpE-LnE of inhibiting Candida virulence factors was assessed with previously reported methods. The effect of ZpE-LnE and of ZpE or LnE alone on cell viability was determined on human hepatoma cells line Huh7., [Results] ZpE-Ln E was fungicidal killing C. albicans in a shorter time than amphotericin B and produced malformations in S. pombe cells. ZpE-LnE showed to bind to ergosterol but not to inhibit any step of the ergosterol biosynthesis. ZpE-LnE showed a low or moderate capacity of inhibiting GS and ChS. Regarding inhibition of virulence factors, ZpE-LnE significantly decreased the capacity of adhesion to eukaryotic buccal epithelial cells (BECs), did not inhibit the germ tube formation and inhibited the secretion of phospholipases and proteinases but not of haemolysins. ZpE-LnE demonstrated very low toxicity on Huh7 cells, much lower than that each extract alone., [Conclusion] The fungicidal properties of ZpE-LnE against C. albicans, its dual mechanism of action targeting the fungal membrane's ergosterol as well as the cell wall, its capacity of inhibiting several important virulence factors added to its low toxicity, make ZpE-LnE a good candidate for the development of a new antifungal bi-Herbal Medicinal Product.

Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model.IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic condi

Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model.IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic condi

Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model.IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic condi

Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model.IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic condi

Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model. IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic cond, Funding Agencies:J. C. Kempes Minnes StipendiefondArneska Stiftelse

PREMISE OF THE STUDY: Lichen-forming fungi produce diverse vegetative tissues, some closely resembling those of plants. Yet it has been repeatedly affirmed that none is a true parenchyma, in which cellular compartments are subdivided from all adjacent neighbors by cross walls adjoining older cross walls. METHODS: Using transmission electron microscopy (TEM), we tested this assumption by examining patterns of septum formation in the parenchyma-like cortex of three lichens of different phylogenetic affinities: Sticta canariensis, Leptogium cyanescens, and Endocarpon pusillum. KEY RESULTS: In the cortex of all three lichens, new septa adjoined perpendicularly or obliquely to previous septa. Septal walls possessed an electron-transparent core (median) layer covered on both sides by layers of intermediate electron density. At septal junctures, the core layer of the newer septum was not continuous with that of the older septum. Amorphous, electron-dense material often became deposited in the core region of older septal walls, and the septum gradually delaminated along its median into what could then be recognized as the distinct walls of neighboring cells. However, cells maintained continuity at pores, where adjacent remnants of the electron-transparent core layer suggested septal partition rather than secondary establishment of a lateral wall connection via anastomosis. CONCLUSIONS: Although fungal tissues first arise by the coalescence of filaments early in lichen ontogeny, the mature cortical tissues of some lichens are comparable to true parenchyma in the unrestricted orientation of their septal cross walls and the resulting ontogenetic relationship among neighboring cell compartments.

The postharvest fungal pathogensPenicillium digitatum, P. italicumand P. expansumare an in-creasing problem for the Mediterranean orchards and fruit industry. This study was designed to gain knowl-edge on factors affecting susceptibility of Penicilliumspp. to antimicrobial peptides (AMPs) as new antifungal compounds for plant protection. The previously characterized PAF26 is a novel penetratin-type AMP with ac-tivity against phytopathogenic fungi. Comparative analyses were conducted on the sensitivity of Penicilliumspp. to PAF26, to the cytolytic peptide melittin and to other antimicrobials. The research included microscopic observations, chitin quantification, virulence assays on citrus and apple fruits, and molecular phylogenetic re-lationships within Penicillium isolates from citrus fruit. Virulence analysis and phylogenetic reconstruction confirmed the host specificity and monophyletic origin for P. digitatum, contrary to the closely-related species P. expansumand P. italicum. A parallelism was found between sensitivity to PAF26 of Penicilliumisolates and to the chitin dye calcofluor white (CFW). No such correlation was found between sensitivity to PAF26 and to the membrane perturbing compound SDS or the oxidizing agent H2O2. Microscopy studies showed that mycelium and conidia from the PAF26-sensitive fungi were also prone to CFW staining, but no direct correlation with the mycelial chitin content was found. The data are consistent with the fact that fungal cell walls influence the outcome of the interaction of AMPs with fungi, and that PAF26 is more active towards Penicilliumcitrus fruit pathogens. In this context, CFW could help both to elucidate AMPs mode of action and in studies of the mechanisms of virulence and host specificity within Penicillium spp.

The goal of this project is extraction of chitosan optimally by surveys of temperature changesalong with 1% Sulfuric acid utilization. Microbial chitosan is isolated as a bio-componentfrom cell wall of two kinds of Zygomycetes by some extraction methods. This projectcompares ability of two type Fungi (R. pulusilus and M.indicus) from Zygomycets forproduction of chitosan.To extract of chitosan is a combinational method with 40 %( w/w) Hydroxyl sodium for celldisruption and diluted Sulfuric acid (1% w/v) for chitosan extraction from cell wall as majorchemical components. 40% NaOH is used to get different degrees of deacetylation (DD) fromchitin for chitosan. In addition, it is examined 1% Sulfuric acid in a combination oftemperature factor changes. It is needed dialysis for chitosan purification from bondedphosphate groups. Standard curves of acetic acid experiences for DD and water phosphatedetermination were accomplished.It has resulted if degree of deacetylated chitin is about 50%; it has an average lost more than50% in 1% (v/v) Sulfuric acid, hence less recovery as a no privilege that it can be relapsed byacetone in chitosan solution. Factor of temperate in same times shows important effect onextraction yield of chitosan by 1% Sulfuric acid. Extracted chitosan in 120℃ has DD about50%. Absolutely, its solubility will be more and it needs to an intricate solution for separationof chitosan from phosphate bonds as a major impurity by dialysis, but in 90℃, DD of chitosanis more with less solubility in water.Between two Fungi, in experienced temperatures, hence, R. pulusilus has more recovery about0.87/AIM (g/g) in 90℃, which have more much DD than 50%, and M.indicus has 0.79/AIM(g/g) in 120℃ that it has DD about 50%.

The goal of this project is extraction of chitosan optimally by surveys of temperature changesalong with 1% Sulfuric acid utilization. Microbial chitosan is isolated as a bio-componentfrom cell wall of two kinds of Zygomycetes by some extraction methods. This projectcompares ability of two type Fungi (R. pulusilus and M.indicus) from Zygomycets forproduction of chitosan.To extract of chitosan is a combinational method with 40 %( w/w) Hydroxyl sodium for celldisruption and diluted Sulfuric acid (1% w/v) for chitosan extraction from cell wall as majorchemical components. 40% NaOH is used to get different degrees of deacetylation (DD) fromchitin for chitosan. In addition, it is examined 1% Sulfuric acid in a combination oftemperature factor changes. It is needed dialysis for chitosan purification from bondedphosphate groups. Standard curves of acetic acid experiences for DD and water phosphatedetermination were accomplished.It has resulted if degree of deacetylated chitin is about 50%; it has an average lost more than50% in 1% (v/v) Sulfuric acid, hence less recovery as a no privilege that it can be relapsed byacetone in chitosan solution. Factor of temperate in same times shows important effect onextraction yield of chitosan by 1% Sulfuric acid. Extracted chitosan in 120℃ has DD about50%. Absolutely, its solubility will be more and it needs to an intricate solution for separationof chitosan from phosphate bonds as a major impurity by dialysis, but in 90℃, DD of chitosanis more with less solubility in water.Between two Fungi, in experienced temperatures, hence, R. pulusilus has more recovery about0.87/AIM (g/g) in 90℃, which have more much DD than 50%, and M.indicus has 0.79/AIM(g/g) in 120℃ that it has DD about 50%.

The presence of cell walls makes plants immobile but fungi (defined as a separate kingdom, Whittaker 1969) have evolved as organisms which, despite the presence of cell wal

Aspergillus (A.) and mucormycetes species cause severe infections in immunocompromised patients. To understand pathomechanisms and antifungal defence in more detail we studied the role of platelets and complement, which are two important innate immunity elements. Recent own studies showed that the fungal polysaccharide galactosaminogalactan (GAG) affects platelets both in its hyphal-bound and in its secreted form. We aimed to further characterize the effect of GAG on platelets in Aspergillus and mucormycetes species both in vitro and in vivo and compare the study with Candida species. Human platelets were incubated with hyphae, yeast or culture supernatants (SN) of various Aspergillus, Candida and mucormycetes isolates respectively. Platelet binding to fungi, platelets activation and complement deposition on platelets were studied by flow cytometry, confocal laser microscopy, and scanning electron microscopy. Positive and negative consequences of platelet activation and opsonisation were also studied by using above mentioned methods. Incubation of platelets with A.fumigatus and A.flavus SN resulted in deposition of secreted fungal material on the platelet surface whereas no deposition was obvious when incubating the platelets with SN of mucormycetes. This deposition of fungal material correlated with expression of GAG and platelet activation by A.fumigatus and A.flavus. Human and mice platelets were capable of significantly adhere to yeast cells of all tested Candida species. Despite the significant platelet-Candida interaction no significant platelet activation was noticed, however supernatant from all the tested species were moderately induced platelets activation. Other GAG effects on the platelets included the deposition of complement factor C3 and the formation of the C5b-9 complex on the platelet surface. A perfect correlation between presence of GAG and platelet activation/opsonisation could be underlined by the comparison with different Aspergillus, Aspergillus (A.) and mucormycetes species cause severe infections in immunocompromised patients. To understand pathomechanisms and antifungal defence in more detail we studied the role of platelets and complement, which are two important innate immunity elements. Recent own studies showed that the fungal polysaccharide galactosaminogalactan (GAG) affects platelets both in its hyphal-bound and in its secreted form. We aimed to further characterize the effect of GAG on platelets in Aspergillus and mucormycetes species both in vitro and in vivo and compare the study with Candida species. Human platelets were incubated with hyphae, yeast or culture supernatants (SN) of various Aspergillus, Candida and mucormycetes isolates respectively. Platelet binding to fungi, platelets activation and complement deposition on platelets were studied by flow cytometry, confocal laser microscopy, and scanning electron microscopy. Positive and negative consequences of platelet activation and opsonisation were also studied by using above mentioned methods. Incubation of platelets with A.fumigatus and A.flavus SN resulted in deposition of secreted fungal material on the platelet surface whereas no deposition was obvious when incubating the platelets with SN of mucormycetes. This deposition of fungal material correlated with expression of GAG and platelet activation by A.fumigatus and A.flavus. Human and mice platelets were capable of significantly adhere to yeast cells of all tested Candida species. Despite the significant platelet-Candida interaction no significant platelet activation was noticed, however supernatant from all the tested species were moderately induced platelets activation. Other GAG effects on the platelets included the deposition of complement factor C3 and the formation of the C5b-9 complex on, Hemalata Deshmukh, Dissertation Medical University of Innsbruck 2018

Aspergillus (A.) and mucormycetes species cause severe infections in immunocompromised patients. To understand pathomechanisms and antifungal defence in more detail we studied the role of platelets and complement, which are two important innate immunity elements. Recent own studies showed that the fungal polysaccharide galactosaminogalactan (GAG) affects platelets both in its hyphal-bound and in its secreted form. We aimed to further characterize the effect of GAG on platelets in Aspergillus and mucormycetes species both in vitro and in vivo and compare the study with Candida species. Human platelets were incubated with hyphae, yeast or culture supernatants (SN) of various Aspergillus, Candida and mucormycetes isolates respectively. Platelet binding to fungi, platelets activation and complement deposition on platelets were studied by flow cytometry, confocal laser microscopy, and scanning electron microscopy. Positive and negative consequences of platelet activation and opsonisation were also studied by using above mentioned methods. Incubation of platelets with A.fumigatus and A.flavus SN resulted in deposition of secreted fungal material on the platelet surface whereas no deposition was obvious when incubating the platelets with SN of mucormycetes. This deposition of fungal material correlated with expression of GAG and platelet activation by A.fumigatus and A.flavus. Human and mice platelets were capable of significantly adhere to yeast cells of all tested Candida species. Despite the significant platelet-Candida interaction no significant platelet activation was noticed, however supernatant from all the tested species were moderately induced platelets activation. Other GAG effects on the platelets included the deposition of complement factor C3 and the formation of the C5b-9 complex on the platelet surface. A perfect correlation between presence of GAG and platelet activation/opsonisation could be underlined by the comparison with different Aspergillus, Aspergillus (A.) and mucormycetes species cause severe infections in immunocompromised patients. To understand pathomechanisms and antifungal defence in more detail we studied the role of platelets and complement, which are two important innate immunity elements. Recent own studies showed that the fungal polysaccharide galactosaminogalactan (GAG) affects platelets both in its hyphal-bound and in its secreted form. We aimed to further characterize the effect of GAG on platelets in Aspergillus and mucormycetes species both in vitro and in vivo and compare the study with Candida species. Human platelets were incubated with hyphae, yeast or culture supernatants (SN) of various Aspergillus, Candida and mucormycetes isolates respectively. Platelet binding to fungi, platelets activation and complement deposition on platelets were studied by flow cytometry, confocal laser microscopy, and scanning electron microscopy. Positive and negative consequences of platelet activation and opsonisation were also studied by using above mentioned methods. Incubation of platelets with A.fumigatus and A.flavus SN resulted in deposition of secreted fungal material on the platelet surface whereas no deposition was obvious when incubating the platelets with SN of mucormycetes. This deposition of fungal material correlated with expression of GAG and platelet activation by A.fumigatus and A.flavus. Human and mice platelets were capable of significantly adhere to yeast cells of all tested Candida species. Despite the significant platelet-Candida interaction no significant platelet activation was noticed, however supernatant from all the tested species were moderately induced platelets activation. Other GAG effects on the platelets included the deposition of complement factor C3 and the formation of the C5b-9 complex on, Hemalata Deshmukh, Dissertation Medical University of Innsbruck 2018