Modelling biological cell attachment and growth on adherent surfaces

A mathematical model, in the form of an integro-partial differential equation, is presented to describe the dynamics of cells being deposited, attaching and growing in the form of a monolayer across an adherent surface. The model takes into account that the cells suspended in the media used for the seeding have a distribution of sizes, and that the attachment of cells restricts further deposition by fragmenting the parts of the domain unoccupied by cells. Once attached the cells are assumed to be able to grow and proliferate over the domain by a process of infilling of the interstitial gaps; it is shown that without cell proliferation there is a slow build up of the monolayer but if the surface is conducive to cell spreading and proliferation then complete coverage of the domain by the monolayer can be achieved more rapidly. Analytical solutions of the model equations are obtained for special cases, and numerical solutions are presented for parameter values derived from experiments of rat mesenchymal stromal cells seeded onto thin layers of collagen-coated polyethylene terephthalate electrospun fibers. The model represents a new approach to describing the deposition, attachment and growth of cells over adherent surfaces, and should prove useful for studying the dynamics of the seeding of biomaterials.     

A novel method for identifying hydrophobicity on fungal surfaces

Fungal surface hydrophobicity has many ecological functions and water contact angles measurement is a direct and simple approach for its characterization. The objective of this study was to evaluate if in-vitro growth conditions coupled with versatile image analysis allows for more accurate fungal contact angle measurements. Fungal cultures were grown on agar slide media and contact angles were measured utilizing a modified microscope and digital camera setup. Advanced imaging software was adopted for contact angle determination. Contact angles were observed in hydrophobic, hydrophilic and a newly created chronoamphiphilic class containing fungi taxa with changing surface hydrophobicity. Previous methods are unable to detect slight changes in hydrophobicity, which provide vital information of hydrophobicity expression patterns. Our method allows for easy and efficient characterization of hydrophobicity, minimizing disturbance to cultures and quantifying subtle variation in hydrophobicity.     

Permeabilization method for in-situ investigation of fungal conidia on surfaces

Aims:  4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in-situ hybridization (FISH) show great potential for the detection of fungal conidia, also conserving the spatial architecture of their colonization. These investigations are often greatly hampered by the complicated wall structure of many fungal taxa. The aim of the present study was to develop an efficient permeabilization strategy for both DAPI staining and the FISH technique, applicable to various fungal species and maintaining their relationships with surfaces.
Methods and Results:  We compared different DAPI staining permeabilization strategies based on alcohol dehydration, surfactants and osmotic shock, tested with Aspergillus niger conidia. Among four permeabilization methods leading to a strong DAPI signal, only one, based on Triton X-100, EDTA and β-mercaptoethanol followed by hyperosmotic stress, appeared suitable for FISH investigation and was successfully applied to an additional 10 fungal taxa and three environmental samples.
Conclusions:  The effective permeabilization method, which employed a combination of surfactant and osmotic strategies, was successfully applied as preliminary step in both DAPI staining and the FISH protocol.
Significance and Impact of the Study:  The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.     

仓储烟叶表面真菌群落组成分析

利用高通量测序平台,对贮存于贵阳库（GY）、坛厂库（TC）和紫云库（ZY）的云南保山C3F（2013）烟叶样品进行宏基因组水平的ITS1基因测序分析,以期揭示陈化烟叶表面真菌群落组成、分布格局及功能分组,探讨化学因子对其结构的影响。结果显示,从15个样品中共检测到了1 173 560条有效序列,包括4个门、140个属;贵阳库优势属为耐干霉菌属Xeromyces,坛厂库优势属为红酵母属Rhodotorula,紫云库优势属为耐干霉菌属Xeromyces;坛厂库优势菌群及变化趋势与贵阳库和紫云库存在差异;真菌群落以腐生营养型和病理营养型为主,分布有潜在人体病原真菌;有机碳、总磷、总钾对真菌群落变化影响显著（P<0.05）。真菌群落组成和格局分布受存储环境、陈化时间及烟叶化学元素的共同影响。     

White-rot fungal growth on sugarcane lignocellulosic residue

Summary Twelve white-rot fungi were grown in solid state culture on sugarcane chips previously fermented by yeast employing the EX-FERM process. The lignocellulosic sugarcane residue had 12.5% permanganate lignin and 81.3% holocellulose. After 5 to 6 weeks at 20° C, all fungi produced a solid residue which had a lower in vitro dry matter enzymatic digestibility than the original bagasse, with the exception of Coriolus versicolor which showed a slight increase of 0.6 units. Four fungi produced a residue with higher soluble solids than the original sample. Lignin losses were rather similar for all fungi tested, an average value of 38.64% of the original value was obtained. About the same amount of hemicellulose was degreaded, 32.22%. Most fungi showed a preference for hemicellulose hydrolysis over cellulose degradation. The two fungi that showed greater cellulolytic activity were Sporotrichum pulverulentum and Dichomitus squalens. No appreciable dry matter losses were detected for Agrocybe aergerita and Flammulina velutipes.     

Modelling and predicting fungal distribution patterns using herbarium data

Aim The main aims of this study are: (1) to test if temperature and related parameters are the primary determinants of the regional distribution of macrofungi (as is commonly recognized for plants); (2) to test if the success of modelling fungal distribution patterns depends on species and distribution characteristics; and (3) to explore the potential of using herbarium data for modelling and predicting fungal species’ distributions. Location The study area, Norway, spans 58–71° N latitude and 4–32° E longitude, and embraces extensive ecological gradients in a small area. Methods The study is based on 1020 herbarium collections of nine selected species of macrofungi and a set of 75 environmental predictor variables, all recorded in a 5 × 5‐km grid covering Norway. Primarily, generalized linear model (GLM; logistic regression) analyses were used to identify the environmental variables that best accounted for the species’ recorded distributions in Norway. Second, Maxent analyses (using variables identified by GLM) were used to produce predictive potential distribution maps for these species. Results Variables relating to temperature and radiation were most frequently included in the GLMs, and between 24.8% and 59.8% of the variation in single‐species occurrence was accounted for. The fraction of variation explained by the GLMs ranged from 41.6% to 59.8% for species with restricted distributions, and from 24.8% to 39.3% for species with widespread/scattered and intermediate distributions. The two‐step procedure of GLM followed by Maxent gave predictions with very high values for the area under the curve (0.927–0.997), and maps of potential distribution were generally credible. Main conclusions We show that temperature is a key factor governing the distribution of macrofungi in Norway, indicating that fungi may respond strongly to global warming. We confirm that modelling success depends partly on species and distribution characteristics, notably on how the distribution relates to the extent of the study area. Our study demonstrates that the combination of GLM and Maxent may be a fruitful approach for biogeography. We conclude that herbarium data improve insight into factors that control the distributions of fungi, of particular value for research on fleshy fungi (mushrooms), which have largely cryptic life cycles.     

Quantitation of microbial growth on surfaces

An equation describing the initial phases of microbial surface colonization is presented. Simultaneous microbial attachment and growth are considered as the primary components of colonization. A table is given that permits determination of growth rate from the density and distribution of cells present on surfaces after incubation in situ. Other methods used to calculate microbial growth rate on surfaces are evaluated. The new procedure is more accurate and less time consuming than those used previously. Published data on microbial surface colonization more closely follow the proposed colonization equation than the exponential growth equation, which overestimates the growth rate.     

The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces

When fungi grow on plant or insect surfaces coated with wax polyesters that protect against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Aerial structures such as hyphae and conidiophores are coated with hydrophobins, which are surface-active proteins involved in adhesion to hydrophobic surfaces. When the industrial fungus Aspergillus oryzae was cultivated in a liquid medium containing the biodegradable polyester polybutylene succinate-coadipate (PBSA), the rolA gene encoding hydrophobin RolA was highly transcribed. High levels of RolA and its localization on the cell surface in the presence of PBSA were confirmed by immunostaining. Under these conditions, A. oryzae simultaneously produced the cutinase CutL1, which hydrolyses PBSA. Pre-incubation of PBSA with RolA stimulated PBSA degradation by CutL1, suggesting that RolA bound to the PBSA surface was required for the stimulation. Immunostaining revealed that PBSA films coated with RolA specifically adsorbed CutL1. Quartz crystal microbalance analyses further demonstrated that RolA attached to a hydrophobic sensor chip specifically adsorbed CutL1. Circular dichroism spectra of soluble-state RolA and bound RolA suggested that RolA underwent a conformational change after its adsorption to hydrophobic surfaces. These results suggest that RolA adsorbed to the hydrophobic surface of PBSA recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis. A fluorescence recovery after photobleaching experiment on PBSA films coated with FITC-labelled RolA suggested that RolA moves laterally on the film. We discuss the novel molecular functions of RolA with regard to plastic degradation.     

Permeabilization of fungal membranes by plant defensins inhibits fungal growth

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.     

Modelling fungal solid-state fermentation: the role of inactivation kinetics

The theoretical mathematical models described in this paper are used to evaluate the effects of fungal biomass inactivation kinetics on a non-isothermal tray solid-state fermentation (SSF). The inactivation kinetics, derived from previously reported experiments done under isothermal conditions and using glucosamine content to represent the amount of biomass, are described in different ways leading to four models. The model predictions show only significant effects of inactivation kinetics on temperature and biomass patterns in the tray SSF after long fermentation periods. The models in which inactivation is triggered by low specific growth rates can predict restricted biomass evolution in combination with a fast temperature increase followed by a slower temperature decrease. Such inactivation might occur when substrate is limiting or products are formed in toxic concentrations. Temperature is predicted to be the key parameter. Oxygen concentration is predicted to become limiting only at high heat conduction and low oxygen diffusion rates. Desiccation of the substrate is predicted not to occur.     

Oriented Schwann cell growth on microgrooved surfaces

Silicon wafers bearing microgrooved surfaces with various groove width, spacing, and depth were fabricated using microlithography. The orientation of rat Schwann cells along the direction of the grooves was measured at 24 h after seeding the cells. When the width/spacing of the grooves was fixed at 10/10 microm, the mean percentage of aligned cells was 12% for grooves of 0.5 microm depth, 15% for those of 1 microm depth, and 26% for those of 1.5 microm depth (P < 0.05). When the depth of grooves was fixed at 1.5 microm, the mean percentage of aligned cells increased from 26% for width/spacing 10/10 microm, to 33% for 10/20 microm or 20/10 microm, and up to 41% for 20/20 microm (P < 0.05). On the surface with grooves of width/spacing/depth = 20/20/1.5 microm and modified by laminin, the alignment at 24 h approached 60%, versus 51% for collagen-coated surface and 41% for uncoated surface (P < 0.05). At 48 h after seeding, about 66% of the cells were aligned on the above laminin-modified surface. The groove depth influenced orientation of Schwann cells significantly. The cell alignment on 20/20/3 microm microgrooved poly(D,L-lactide-co-glycolide) 90:10 (PLGA) surfaces transferred from silicon reached 72% at 48 h and 92% at 72 h (P < 0.05). Coating this surface with laminin enhanced cell alignment only in short term (67% vs. 62% at 24 h, P < 0.05). The cell alignment guided by surface microgrooves was time dependent.     

Modelling Si-N-limited growth of diatoms

A mechanistic model for silicon (Si) physiology is developed,interfaced with a model of nitrogen (N) physiology, which iscapable of simulating the major documented facets of Si–Nphysiology in diatoms. The model contains a cell cycle componentthat is involved in regulating the timing of the synthesis ofvalves, girdles and setae. In addition to reproducing the timingof diatom cell division within a light–dark cycle, themodel simulates the following features seen in real diatoms.Synthesis of valves only occurs during G₂ interphase and M,while the girdles and (if appropriate) setae are synthesizedduring G₁. Si stress alone results in a loss of setae, followedby a thinning of the valves in successive generations untila minimum Si cell quota is attained. After this point, the durationof G₂ increases and growth is Si limited. Concurrently, thecarbon (C) cell quota increases, offering the capability tosimulate the documented increase in sinking rates with Si stress.N stress alone results in an increase in the duration of G₁and G₂ interphases, and high Si cell quotas. From this complexmodel, which must be run for arrays of subpopulations to simulatenon-synchronous growth, a simpler model is developed. This iscapable of reproducing similar growth dynamics, although withno reference to component parts of the frustule. When alliedto a photoacclimative submodel, a prediction of the model isthat diatoms starved of Si will release increased amounts ofdissolved organic C because cell growth is halted more rapidlythan the photosystems can be degraded.     

Modelling the bacterial growth/no growth interface

A logistic regression model is proposed which enables one to model the boundary between growth and no growth for bacterial strains in the presence of one or more growth controlling factors such as temperature, pH and additives such as salt and sodium nitrite. The form of the expression containing the growth limiting factors may be suggested by a kinetic model, while the response at a given combination of factors may either be presence/absence (i.e. growth/no growth) or probabilistic (i.e. r successes in n trials). The approach described represents an integration of the probability and kinetic aspects of predictive microbiology, and a unification of predictive microbiology and the hurdle concept. The model is illustrated using data for Shigella flexneri.     

Examination of fungal growth and aflatoxin production on marihuana

Under favorable growth conditions,Aspergillus flavus andA. parasiticus produced aflatoxins on marihuana. Cultures ofA. flavus ATCC 15548 produced both aflat oxin B₁(AFB₁) and G₁(AFG₁). The production of AFG₁ was substantially greater than that of AFB₁. Cultures ofA. flavus NRRL 3251 andA. parasiticus NRRL 2999 produced only AFB₁. All natural flora cultures tested negative for aflatoxins. NoAspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 g/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.     

Influence of litterbags on growth of fungal vegetative structures

Summary This study reports a test of the hypothesis that mesh litter bags restrict access of vegetative fungal structures to confined substrate. Unconfined birchwood swab sticks lost more dry weight during a field incubation than confined ones. Samples in fine mesh bags lost more weight than those in coarse mesh bags. Significantly higher numbers of mycelial strands and rhizomorphs were found on unconfined samples. The number of fungal structures was significantly correlated with weight loss of individual samples. It is suggested that the inhibitory effect is most significant in environments where higher fungi are the dominant decomposing organisms.     

Effect of fungal staling growth substances on colony interaction

Summary Effect of staling growth substances on the colony interaction in between some dominant rhizosphere fungi andSclerotium rolfsii Sacc. a root pathogen ofLens esculantum Moench. was examinedin vitro. Experimental findings depict that staling growth substances diffused in the agar created the change in the pattern of colony interaction. Measurement of growth inhibition of colony interaction was assessed with the help of a proposed model.     

Gliotoxin effects on fungal growth: mechanisms and exploitation

Although initially investigated for its antifungal properties, little is actually known about the effect of gliotoxin on Aspergillus fumigatus and other fungi. We have observed that exposure of A. fumigatus to exogenous gliotoxin (14 μg/ml), under gliotoxin-limited growth conditions, results in significant alteration of the expression of 27 proteins (up- and down-regulated >1.9-fold; p<0.05) including de novo expression of Cu, Zn superoxide dismutase, up-regulated allergen Asp f3 expression and down-regulated catalase and a peroxiredoxin levels. Significantly elevated glutathione GSH levels (p<0.05), along with concomitant resistance to diamide, were evident in A. fumigatus ΔgliT, lacking gliotoxin oxidoreductase, a gliotoxin self-protection gene. Saccharomyces cerevisiae deletents (Δsod1 and Δyap1) were hypersensitive to exogenous gliotoxin, while Δgsh1 was resistant. Significant gliotoxin-mediated (5 μg/ml) growth inhibition (p<0.001) of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, Cochliobolus heterostrophus and Neurospora crassa was also observed. Growth of Aspergillus flavus, Fusarium graminearum and Aspergillus oryzae was significantly inhibited (p<0.001) at gliotoxin (10 μg/ml), indicating differential gliotoxin sensitivity amongst fungi. Re-introduction of gliT into A. fumigatus ΔgliT, at a different locus (ctsD; AFUA_4G07040, an aspartic protease), with selection on gliotoxin, facilitated deletion of ctsD without use of additional antibiotic selection markers. Absence of ctsD expression was accompanied by restoration of gliT expression, and resistance to gliotoxin. Thus, we propose gliT/gliotoxin as a useful selection marker system for fungal transformation. Finally, we suggest incorporation of gliotoxin sensitivity assays into all future fungal functional genomic studies.     

Cellular automata simulations of fungal growth on solid substrates

Growth of filamentous fungi on the surface of cereal grains is a critical aspect of solid substrate fermentation (SSF). Numerous mathematical models have been developed to describe various aspects of fungal growth in SSF. These models consider hyphal geometry and nutrient availability as determinants of colony morphology and fungal physiological state. This work describes the use of cellular automata (CA) as an alternative method of modeling fungal growth. CA models reliant on a very limited set of rules or "knowledge base" display a rich array of behaviors that mimic fungal growth. By incorporating probablistic growth rules into CA models, colony characteristics such as biomass accumulation rate, colony radial growth rate, mycelial density and fungal differentiation are readily generated.     

Biomaterials surfaces capable of resisting fungal attachment and biofilm formation

Microbial attachment onto biomedical devices and implants leads to biofilm formation and infection; such biofilms can be bacterial, fungal, or mixed. In the past 15 years, there has been an increasing research effort into antimicrobial surfaces but the great majority of these publications present research on bacteria, with some reports also testing resistance to fungi. Very few studies have focused exclusively on antifungal surfaces. However, with increasing recognition of the importance of fungal infections to human health, particularly related to infections at biomaterials, it would seem that the interest in antifungal surfaces is disproportionately low. In studies of both bacteria and fungi, fungi tend to be the minor focus with hypothesized antibacterial mechanisms of action often generalized to also explain the antifungal effect. Yet bacteria and fungi represent two Distinct biological Domains and possess substantially different cellular physiology and structure. Thus it is questionable whether these generalizations are valid. Here we review the scientific literature focusing on surface coatings prepared with antifungal agents covalently attached to the biomaterial surface. We present a critical analysis of generalizations and their evidence. This review should be of interest to researchers of “antimicrobial” surfaces by addressing specific issues that are key to designing and understanding antifungal biomaterials surfaces and their putative mechanisms of action.