Lateral fungal spore movement inside a simulated wall

Moisture inside walls can facilitate mold growth if left untreated. Once spores become airborne they may interact with pressures inside walls. Two laboratory experiments were conducted to determine if airborne spores have the potential to migrate laterally inside walls with and without wiring installations. A simulated wall was fabricated, and Penicillium chrysogenum spores were aerosolized into a distant stud bay and an adjacent stud bay. The wall was subjected to a typical indoor pressure. Spore levels inside the bays were sampled, and a total of 36 trials (n = 36) were conducted. Results of Kruskal–Wallis tests revealed that spore levels inside the sampling bay and the distant bay with wiring installations were not significantly different. Spore levels inside the sampling bay were significantly lower than the adjacent bay without wiring installations (P < 0.05). The findings of the study suggest airborne fungal spores have the potential to move laterally inside walls.     

Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots

Background

Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the “in-cell western” (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC₂₀) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.

Methodology/Principal Findings

ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey®) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC₂₀ phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.

Conclusion/Significance

ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required.     

Effect of electrical charges and fields on injury and viability of airborne bacteria

In this study, the effects of the electric charges and fields on the viability of airborne microorganisms were investigated. The electric charges of different magnitude and polarity were imparted on airborne microbial cells by a means of induction charging. The airborne microorganisms carrying different electric charge levels were then extracted by an electric mobility analyzer and collected using a microbial sampler. It was found that the viability of Pseudomonas fluorescens bacteria, used as a model for sensitive bacteria, carrying a net charge from 4100 negative to 30 positive elementary charges ranged between 40% and 60%; the viability of the cells carrying >2700 positive charges was below 1.5%. In contrast, the viability of the stress-resistant spores of Bacillus subtilis var. niger (used as simulant of anthrax-causing Bacillus anthracis spores when testing bioaerosol sensors in various studies), was not affected by the amount of electric charges on the spores. Because bacterial cells depend on their membrane potential for basic metabolic activities, drastic changes occurring in the membrane potential during aerosolization and the local electric fields induced by the imposed charges appeared to affect the sensitive cells' viability. These findings facilitate applications of electric charging for environmental control purposes involving sterilization of bacterial cells by imposing high electric charges on them. The findings from this study can also be used in the development of new bioaerosol sampling methods based on electrostatic principles.     

Integrated Constructed Wetlands (ICW) for livestock wastewater management

Social, economic and environmental coherence is sought in the management of livestock wastewater. Wetlands facilitate the biogeochemical processes that exploit livestock wastewater and provide opportunities to achieve such coherence and also to deliver on a range of ecosystem services. The Integrated Constructed Wetland (ICW) concept integrates three inextricably linked objectives: water quantity and quality management, landscape-fit to improve aesthetic site values and enhanced biodiversity. The synergies derived from this explicit integration allow one of the key challenges for livestock management to be addressed. An example utilizing twelve ICW systems from a catchment on the south coast of Ireland demonstrates that over an eight year period mean reduction of total and soluble phosphorus (molybdate reactive phosphorus) exceeded 95% and the mean removal of ammonium-N exceeded 98%. This paper reviews evidence regarding the capacity of ICWs to provide a coherent and sustainable alternative to conventional systems.     

Inverted Current–Voltage Hysteresis in Mixed Perovskite Solar Cells: Polarization,Energy Barriers,and Defect Recombination

Organic‐inorganic metal halide perovskite solar cells show hysteresis in their current–voltage curve measured at a certain voltage sweep rate. Coinciding with a slow transient current response, the hysteresis is attributed to a slow voltage‐driven (ionic) charge redistribution in the perovskite solar cell. Thus, the electric field profile and in turn the electron/hole collection efficiency become dependent on the biasing history. Commonly, a positive prebias is beneficial for a high power‐conversion efficiency. Fill factor and open‐circuit voltage increase because the prebias removes the driving force for charge to pile‐up at the electrodes, which screen the electric field. Here, it is shown that the piled‐up charge can also be beneficial. It increases the probability for electron extraction in case of extraction barriers due to an enhanced electric field allowing for tunneling or dipole formation at the perovskite/electrode interface. In that case, an inverted hysteresis is observed, resulting in higher performance metrics for a voltage sweep starting at low prebias. This inverted hysteresis is particularly pronounced in mixed‐cation mixed‐halide systems which comprise a new generation of perovskite solar cells that makes it possible to reach power‐conversion efficiencies beyond 20%.     

Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling.

Aerobiological monitoring was conducted in an experimental room to aid in the development of standardized sampling protocols for airborne microorganisms in the indoor environment. The objectives of this research were to evaluate the relative efficiencies of selected sampling methods for the retrieval of airborne fungal spores and to determine the effect of human activity on air sampling. Dry aerosols containing known concentrations of Penicillium chrysogenum spores were generated, and air samples were taken by using Andersen six-stage, Surface Air System, Burkard, and depositional samplers. The Andersen and Burkard samplers retrieved the highest numbers of spores compared with the measurement standard, an aerodynamic particle sizer located inside the room. Data from paired samplers demonstrated that the Andersen sampler had the highest levels of sensitivity and repeatability. With a carpet as the source of P. chrysogenum spores, the effects of human activity (walking or vacuuming near the sampling site) on air sampling were also examined. Air samples were taken under undisturbed conditions and after human activity in the room. Human activity resulted in retrieval of significantly higher concentrations of airborne spores. Surface sampling of the carpet revealed moderate to heavy contamination despite relatively low airborne counts. Therefore, in certain situations, air sampling without concomitant surface sampling may not adequately reflect the level of microbial contamination in indoor environments.     

Analysis of environmental factors and their effects on fungal spores in the atmosphere of a tropical urban area (San Juan,Puerto Rico)

In Puerto Rico, respiratory diseases affect a significant percentage of the population, and it is possible that the environmental factors unique to the island, such as easterly winds, dense vegetation, and high humidity, may increase the concentration of airborne biological particulates that could distress susceptible individuals. As most studies characterized fungal spores and pollen in temperate zones, we wanted to describe their concentrations in a tropical zone. The Allergenco^® (MK3) spore trap was used to monitor airborne spores and pollen concentrations for 12 months starting on May 2005. During the length of the study, silicone grease-coated microscope slides captured 12 impacts daily, which were then stained with calcofluor and observed under the microscope with bright field and ultraviolet light. Atmospheric conditions were recorded with the Precision Weather Station^®. Precipitation, dew point, and relative humidity promote the presence of fungal spores in San Juan throughout the year, with basidiospores being the most frequent. Spore concentrations were higher during the early morning hours all the year around. Wind and gusts were dispersal factors, and humidity and the dew point favored the release of spores into the atmosphere. In contrast to spores, pollens were not found to be affected by any of the meteorological factors and no distinctive patterns were found. Describing how airborne spores and pollens, many of which have allergenic potential, behave in the tropics, such as in San Juan, Puerto Rico, could be used to design preventive measures for the benefit of the sensitized population. Information about the presence and abundance of aeroallergens will assist in the diagnosis of allergic rhinitis and asthma on the island.     

Point mutation in the mouse P2X7 receptor affects intercellular calcium waves in astrocytes

Purinergic P2 receptors and gap junctions are two groups of proteins involved in the transmission of ICWs (intercellular calcium waves) between astrocytes. The extent to which ICWs spread among these glial cells depends on the amount of ATP released, which can occur through membrane channels, as well as other pathways. Our previous studies have shown that the pore-forming P2X₇R (P2X₇ receptor) contributes to the amplification of ICW spread by providing sites of ATP release through Panx1 (Pannexin1) channels. To gain insight into the signal transduction events mediating this response we compared the properties of the P2X₇R–Panx1 complex in astrocytes from a mouse strain (C57Bl/6) containing a naturally occurring point mutation (P451L) in the C-terminus of the P2X₇R to that of non-mutated receptors (Balb/C mice). Electrophysiological, biochemical, pharmacological and fluorescence imaging techniques revealed that the P451L mutation located in the SH3 domain (a Src tyrosine kinase-binding site) of the C-terminus of the P2X₇R attenuates Panx1 currents, ATP release and the distance of ICW spread between astrocytes. Similar results were obtained when using the Src tyrosine inhibitor (PP2) and a membrane-permeant peptide spanning the P451L mutation of the P2X₇R of the C57Bl6 astrocytes. These results support the participation of a tyrosine kinase of the Src family in the initial steps mediating the opening of Panx1 channels following P2X₇R stimulation and in the transmission of calcium signals among astrocytes.     

Resistance of yeast and bacterial spores to high voltage electric pulses

Spores of a yeast, Saccharomyces cerevisiae, and a bacterium, Bacillus subtilis, were exposed to high voltage electric pulses. The viabilities of spores and vegetative cells of the yeast were significantly decreased after the electric pulse treatment, and some of the spores and almost all of the cells were stained red with an agent, phloxine B. On the other hand, (endo) spores of the bacterium were highly resistant to the electric pulses and little decrease in viability was observed, although the viability of vegetative cells was sharply lowered. The results revealed marked structural and/or biochemical differences between eukaryotic and prokaryotic spores.     

Surface modifications by field induced diffusion

By applying a voltage pulse to a scanning tunneling microscope tip the surface under the tip will be modified. We have in this paper taken a closer look at the model of electric field induced surface diffusion of adatoms including the van der Waals force as a contribution in formations of a mound on a surface. The dipole moment of an adatom is the sum of the surface induced dipole moment (which is constant) and the dipole moment due to electric field polarisation which depends on the strength and polarity of the electric field. The electric field is analytically modelled by a point charge over an infinite conducting flat surface. From this we calculate the force that cause adatoms to migrate. The calculated force is small for voltage used, typical 1 pN, but due to thermal vibration adatoms are hopping on the surface and even a small net force can be significant in the drift of adatoms. In this way we obtain a novel formula for a polarity dependent threshold voltage for mound formation on the surface for positive tip. Knowing the voltage of the pulse we then can calculate the radius of the formed mound. A threshold electric field for mound formation of about 2 V/nm is calculated. In addition, we found that van der Waals force is of importance for shorter distances and its contribution to the radial force on the adatoms has to be considered for distances smaller than 1.5 nm for commonly used voltages.     

A stochastic two-dimensional model of intercellular Ca2+ wave spread in glia

We describe a two-dimensional stochastic model of intercellular Ca(2+) wave (ICW) spread in glia that includes contributions of external stimuli, ionotropic and metabotropic P2 receptors, exo- and ecto-nucleotidases, second messengers, and gap junctions. In this model, an initial stimulus evokes ATP and UTP release from a single cell. Agonists diffuse and are degraded both in bulk solution and at cell surfaces. Ca(2+) elevation in individual cells is determined by bound agonist concentrations s and by number and features of P2 receptors summed with that generated by IP(3) diffusing through gap junction channels. Variability of ICWs is provided by randomly distributing a predetermined density of cells in a rectangular grid and by randomly selecting within intervals values characterizing the extracellular compartment, individual cells, and interconnections with neighboring cells. Variability intervals were obtained from experiments on astrocytoma cells transfected to express individual P2 receptors and/or the gap junction protein connexin43. The simulation program (available as Supplementary Material) permits individual alteration of ICW components, allowing comparison of simulations with data from cells expressing connexin43 and/or various P2 receptor subtypes. Such modeling is expected to be useful for testing phenomenological hypotheses and in understanding consequences of alteration of system components under experimental or pathological conditions.     

Investigation of the electrostatic charge of basidiospores of the Phellinus igniarius group

The polarity and magnitude of primary electric charges carried by basidiospores in the airborne state were investigated in living fungal fruiting bodies under natural forest conditions using a portable experimental device designed by the author. The operating principle was the falling of spores in the homogeneous horizontal electric field. The vertical and horizontal components of the trajectories of the spores were determined according to their deposition sites on electrodes (vertical metal plates). Altogether 33 samples of spores were examined for polarity, 10 of these samples (with 10⁴–10⁶ spores per sample) also were used to calculate the mean spore charge-to-mass quotient and the mean spore charge. The detection limits of spore charge-to-mass quotient varied in the range from (4.9±2.3)×10^?5 to (1.36±0.33)×10^?4 C kg^?1. Basidiospores (subglobose, smooth, diameter of 4–6 μm) of the closely related (sibling) species Phellinus alni, P. nigricans, P. populicola and P. tremulae (Hymenochaetales, Basidiomycota) carried positive electrical charges that have mean values from 48 to 305 elementary charges. The intraspecies variation of the spore charge could depend on the natural variation in spore size.     

Airborne fungal spores in the coastal plain of Israel: A preliminary survey

Airborne spores were monitored during the years 1993–1995 in three cities along the coastal plain of Israel: Ramat Gan, Tel Aviv (Ramat Aviv) and Haifa. Seasonal fluctuations in the concentration of airborne spores were recorded. The following genera of fungi were identified:Alternaria, Cladosporium, Coprinus, Curvularia, Drechslera. Diplococcum, Epicoccum, Fusarium, Leptosphaeria, Pithomyces, Puccinia, Sphacelotheca, Stemphylium andUstilago. Unidentified spores were very rare and in negligible numbers. The dominant airborne fungal spores wereCladosporium andAlternaria. The monthly variations in airborne spores, observed among the three cities, seem to be rather minor. The recorded levels of airborne spores were below the concentrations that are accepted as threshold levels for provocation of clinical responses.     

The electrostatic nature of the spore of Pasteuria penetrans, the bacterial parasite of root-knot nematodes

The response of spores of Pasteuria penetrans , the Gram-positive obligate nematode hyperparasite, was studied in a direct current electric field and monitored using a microscope attached to a video recorder apparatus. Fluorescence measurements were performed on the spore's surface using HEXCO as a fluorescent probe. The mobilities of the spores and fluorescence measurements were performed in different salt concentrations and at different pH values. The results showed a significant electronegative potential at the spore surface which was dependent on the pH, salt concentration and valency of the cation present in the electrolyte medium. The results of the fluorescence experiments using HEXCO correlated well with the results obtained from the electrophoretic mobility experiments. A polyclonal antiserum raised to spores of P. penetrans affected the surface charge density and the data presented suggest that electrostatic interactions may be important in the binding of spores to the nematode cuticle. The binding of HEXCO to hydrophobic sites on the spores' surfaces suggests the possibility of other attractive forces also being important in the binding process.     

Rapid evaluation of a protein-based voltage probe using a field-induced membrane potential change

The development of a high performance protein probe for the measurement of membrane potential will allow elucidation of spatiotemporal regulation of electrical signals within a network of excitable cells. Engineering such a probe requires a functional screen of many candidates. Although the glass-microelectrode technique generally provides an accurate measure of a given test probe, throughputs are limited. In this study, we focused on an approach that uses the membrane potential changes induced by an external electric field in a geometrically simple mammalian cell. For quantitative evaluation of membrane voltage probes that rely on the structural transition of the S1–S4 voltage sensor domain and hence have non-linear voltage dependencies, it was crucial to introduce exogenous inwardly rectifying potassium conductance to reduce cell-to-cell variability in resting membrane potentials. Importantly, the addition of the exogenous conductance drastically altered the profile of the field-induced potential. Following a site-directed random mutagenesis and the rapid screen, we identified a mutant of a voltage probe Mermaid, exhibiting positively shifted voltage sensitivity. Due to its simplicity, the current approach will be applicable under a microfluidic configuration to carry out an efficient screen. Additionally, we demonstrate another interesting aspect of the field-induced optical signals, ability to visualize electrical couplings between cells.     

The microbiology of moist barley storage in unsealed silos

The microflora of moist barley grain and whole-crop barley silage stored in top-unloading, unsealed concrete-staved silos on six farms in England depended on the initial water content of the grain (23–58%), method of covering the grain, and the rate at which it was unloaded. Fungi and actinomycetes were fewest when the initial water content was more than 30%, and the grain was covered first with a layer of wilted grass, and then a plastic sheet. During unloading, the uppermost layer of grain remained in good condition provided 7.5 cm was removed daily. With an inefficient top-seal, the top grain heated and became mouldy, as it also did when unloading was slow. As the rate of unloading slowed, heating increased, and a characteristic succession of fungi and actinomycetes developed. With unloading at 7.5 cm/day or more, only yeasts, chiefly Endomycopsis chodatii Wickerham and Hansenula anomala (Hansen) H. & P. Sydow, were abundant, but at slightly slower rates of unloading Penicillium spp. also became common. Both these groups became less common as unloading was slowed further and were replaced, first by Absidia spp. and Mucor pusillus Lindt, then Aspergillus fumigatus Fres., Humicola lanuginosa (Griffon & Maublanc) Bunce, Micropolyspora faeni Cross, Maciver & Lacey, and Thermoactinomyces vulgaris Tsiklinsky as the heating increased. The number of spores (including bacterial cells) that could be removed from samples by blowing air ranged from 0.4–428 times 10⁶/g dry weight of grain. Whole-crop barley silage contained 2.9–132 times 10⁶spores/g dry weight. Similar species were isolated from whole-crop silage as from grain. Little moulding occurred deeper than 30 cm below surface of the grain. Concentrations of airborne spores were estimated periodically during two seasons. There were always more airborne spores than is usual in outdoor air. Without disturbance the silos contained 10⁶-10⁷ spores/m³ air, but when mouldy grain was unloaded concentrations increased to a maximum of 2860times10⁶ spores/m³ air; more than half of these were bacteria and actinomycetes and a quarter Aspergillus flavus Link. Potentially pathogenic fungi and actinomycetes were frequent, particularly when they also occurred in the grain or capping materials for spontaneous heating. Some probably survived in dust deposits and were resuspended during unloading. Airborne spores were frequent around the silos when grain was unloaded and rolled. Workers should wear efficient dust respirators at these times and while inside silos.     

Enhanced photocatalytic inactivation of bacterial spores on surfaces in air

TiO(2) photocatalysis with ultraviolet (UV-A) light has proven to be a highly effective process for complete inactivation of airborne microbes. However, the overall efficiency of the technology needs to be improved to make it more attractive as a defense against bio-terrorism. The present research investigates the enhancement in the rate of destruction of bacterial spores on metal (aluminum) and fabric (polyester) substrates with metal (silver)-doped titanium dioxide and compares it to conventional photocatalysis (TiO(2) P25/+UV-A) and UV-A photolysis. Bacillus cereus bacterial spores were used as an index to demonstrate the enhanced disinfection efficiency. The results indicate complete inactivation of B. cereus spores with the enhanced photocatalyst. The enhanced spore destruction rate may be attributed to the highly oxidizing radicals generated by the doped TiO(2).     

Ex Vivo and In Silico Feasibility Study of Monitoring Electric Field Distribution in Tissue during Electroporation Based Treatments

Magnetic resonance electrical impedance tomography (MREIT) was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment.     

电场对完整青蛙横纹肌收缩机理的椭圆偏振法研究

利用能反映肌纤维内部结构的椭圆偏振参数研究恒定电场,脉冲电场对完整的青蛙横纹肌收缩的影响,研究表明,电场对肌纤维作用后,肌质网释放钙离子,致使肌纤维中蛋白质分子运动后出现收缩效应。实验发现：椭圆偏参数的测量灵敏度和肌小节长度有关,蛋白质运动规律和电场的性质的关系无的差异,只有量的不同。它们的共同特点是,随着电场强度的逐渐增强,其退偏率的变化呈先下降一上升的凹形曲线,这是由于电场引起的肌球蛋白分子极     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.