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Substrate aggregation due to aerial hyphae during discontinuously mixed solid-state fermentation with Aspergillus oryzae: experiments and modeling.

Solid-state fermentation (SSF) is prone to process failure due to channeling caused by evaporative cooling and the formation of an interparticle mycelium network. Mixing is needed to break the mycelium network and to avoid such failure. This study presents the first attempt to quantify and predict the effect of mycelium bonds on particle mixing and vice versa. We developed a novel experimental set-up to measure the tensile strength of hyphal bonds in SSF: Aspergillus oryzae was cultivated between two wheat-dough disks and the tensile strength of the aerial mycelium was measured with a texture analyzer. Tensile strength at different incubation times was related to oxygen consumption, to allow a translation to a rotating drum with A. oryzae cultivated on wheat grain. We performed several discontinuously mixed solid-state fermentations in the drum fermentor and measured the number and size of grain-aggregates remaining after the first mixing action. We integrated data on mycelium tensile strength into a previously developed two-dimensional discrete-particle model that calculates forces acting on individual substrate particles and the resulting radial-particle movements. The discrete-particle model predicted the quantity and size of the aggregates remaining after mixing successfully. The results show that the first mixing event in SSF with A. oryzae is needed to break mycelium to avoid aggregate formation in the grain bed, and not to distribute water added to compensate for evaporation losses, or smooth out temperature gradients.

Aspergillus↗

A novel in vitro cultivation system to produce and isolate soluble factors released from hyphae of arbuscular mycorrhizal fungi.

The Petri plate dual in vitro culture system for arbuscular mycorrhizal fungi (AMF) is ill-suited for the production and isolation of extraradically released soluble factors, thereby preventing the further characterization of such compounds. To overcome this technical bottleneck, we here describe a novel cultivation system using the standard AMF Glomus intraradices-carrot dual culture as a model. This new system is capable of producing soluble factors in copious amounts without any interference from host roots. The greatest advantages of this culture method include ease of handling and reusability of the culture vessel, thus making it a very cost effective system.

Bioreactors↗

Nuclear anarchy: asynchronous mitosis in multinucleated fungal hyphae.

Multinucleated cells are found in diverse contexts and include filamentous fungi, developing insect embryos, skeletal muscle and metastasizing tumor cells. Some multinucleated cells such as those in muscles arise from cell fusion events, but many are formed through specialized cell cycles in which nuclear and cell division are uncoupled. Recent work in the fungus Ashbya gossypii illustrates how unique spatial and temporal regulation of conserved cell cycle regulators directs mitosis in multinucleated cells.

Cell Cycle Proteins↗

Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa.

Previous work has shown that hyphal elongation in the fungus Neurospora crassa requires a tip-high cytosolic Ca2+ gradient. The source of the Ca2+ appears to be intracellular stores as there is no net transplasma membrane Ca2+ flux at the elongating hyphal tip and modification of ion fluxes across the plasma membrane using voltage clamp is without effect on tip growth. To decode the internal mechanisms which generate and maintain the tip-high Ca2+ gradient we first identified calcium regulators which affect hyphal growth and morphology, then determined how they modify cytosolic [Ca2+] and the actin cytoskeleton using fluorescent dyes and confocal microscopy. Cyclopiazonic acid (a known inhibitor of the endoplasmic reticulum calcium ATPase) inhibits growth and increases cytoplasmic [Ca2+] in the basal region 10-25 microm behind the hyphal tip. 2-APB (2-aminoethoxydiphenyl borate, an inhibitor of IP3-induced Ca2+ release) inhibits hyphal elongation and dissipates the tip-high Ca2 gradient 0-10 microm from the tip. Microinjections of the IP3 receptor agonists adenophostin A and IP3 (but not control microinjections of the biologically inactive L-IP3) transiently inhibited growth and induced subapical branches. IP3 microinjections, but not L-IP3, lowered tip-localized [Ca2+] and increased basal [Ca2+]. Even though their effect on [Ca2+] gradients was different, both cyclopiazonic acid and 2-APB disrupted similarly the normal actin pattern at the hyphal apex. Conversely, disruption of actin with latrunculin B dissipated tip-localized Ca2+. We conclude that the tip-high Ca2+ gradient is generated internally by Ca2+ sequestration into endoplasmic reticulum behind the tip and Ca2+ release via an IP3 receptor from tip-localized vesicles whose location is maintained by the actin cytoskeleton.

Actins↗

Comparison of gene expression in trap cells and vegetative hyphae of the nematophagous fungus Monacrosporium haptotylum.

Nematode-trapping fungi enter the parasitic stage by developing specific morphological structures called traps. The global patterns of gene expression in traps and mycelium of the fungus Monacrosporium haptotylum were compared. The trap of this fungus is a unicellular spherical structure called the knob, which develops on the apex of a hyphal branch. RNA was isolated from knobs and mycelium and hybridized to a cDNA array containing probes of 2822 EST clones of M. haptotylum. Despite the fact that the knobs and mycelium were grown in the same medium, there were substantial differences in the patterns of genes expressed in the two cell types. In total, 23.3 % (657 of 2822) of the putative genes were differentially expressed in knobs versus mycelium. Several of these genes displayed sequence similarities to genes known to be involved in regulating morphogenesis and cell polarity in fungi. Among them were several putative homologues for small GTPases, such as rho1, rac1 and ras1, and a rho GDP dissociation inhibitor (rdi1). Several homologues to genes involved in stress response, protein synthesis and protein degradation, transcription, and carbon metabolism were also differentially expressed. In the last category, a glycogen phosphorylase (gph1) gene homologue, one of the most upregulated genes in the knobs as compared to mycelium, was characterized. A number of the genes that were differentially expressed in trap cells are also known to be regulated during the development of infection structures in plant-pathogenic fungi. Among them, a gas1 (mas3) gene homologue (designated gks1), which is specifically expressed in appressoria of the rice blast fungus, was characterized.

Animals↗

Subtle intracorneal findings in inflammatory disorders: hyphae or not?

BACKGROUND: Spongiotic and lichenoid dermatitides are frequently stained with periodic acid-Schiff (PAS) stains to check for the presence of dermatophytes. PAS+ structures without a septate morphology are often seen with lichenoid dermatitides, however, their nature has not been previously characterized. METHODS: Fifteen consecutive biopsies of lichenoid and five spongiotic dermatitides were stained with hematoxylin and eosin (H&E), PAS, and antibodies to CD1a. RESULTS: Twelve of 15 lichenoid and none of the five spongiotic dermatitis showed PAS+ structures in the stratum corneum. Distinct septation or branching was not identified in these PAS+ structures. Eleven of 15 from the lichenoid group, but none from the spongiotic group, showed CD1a+ structures in the stratum corneum. This staining pattern suggests that the intracorneal structures represent the dendritic processes of Langerhans' cells (LCs) within the stratum corneum. CONCLUSIONS: PAS+ and CD1a+ structures are present in the stratum corneum of lichenoid, but not in spongiotic, dermatitis. This study morphologically confirms extension of LC dendrites into the stratum corneum in lichenoid but not in spongiotic dermatitides.

Antigens, CD1↗