To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (approximately 6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 +/- 0.2-fold and V 10.7 +/- 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10 degrees C) reduced SA and V increases of A549 cells to 1.7 +/- 0.3-fold and 4.4 +/- 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously 2+ -dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

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Background:The stoichiometry of pore-forming toxins is frequently unknown because crystal structures do not reflect the active conformations. Results: We used single subunit counting on fluorescently labeled Cry1Aa toxin of Bacillus thuringiensis to follow its oligomerization process. Conclusion: We determined that the final architecture of the pores is tetrameric. Significance: The stochastic analysis introduced permits the application of single subunit counting to dynamic processes such as oligomerization.

Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Förster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven α helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the α3–α4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time course of the donor fluorescence. We found that Cry1Aa is present on both membrane leaflets.

Background: Bioaerosol sampling devices are necessary for the characterization of infectious bioaerosols emitted by naturally-infected hosts with acute respiratory virus infections. Assessment of these devices under multiple experimental conditions will provide insight for device use. Objectives:The primary objective of this study was to assess and compare bioaerosol sampling devices using a) an in vitro, environmentally-controlled artificial bioaerosol system at a range of different RH conditions and b) an in vivo bioaerosol system of influenza virus-infected ferrets under controlled environmental conditions. Secondarily, we also sought to examine the impact of NSAIDs on bioaerosol emission in influenza virus-infected ferrets to address its potential as a determinant of bioaerosol emission. Methods:We examined the performance of low and moderate volume bioaerosol samplers for the collection of viral RNA and infectious influenza virus in vitro and in vivo using artificial bioaerosols and the ferret model of influenza virus infection. The following samplers were tested: the polytetrafluoroethylene filter (PTFE filter), the 2stage National Institute of Occupational Safety and Health cyclone sampler (NIOSH cyclone sampler) and the 6-stage viable Andersen impactor (Andersen impactor). Results:The PTFE filter and NIOSH cyclone sampler collected similar amounts of viral RNA and infectious virus from artificially-generated aerosols under a range of relative humidities (RH). Using the ferret model, the PTFE filter, NIOSH cyclone sampler and the Andersen impactor collected up to 3.66 log 10 copies of RNA/L air, 3.84 log 10 copies of RNA/L air and 6.09 log 10 copies of RNA/L air respectively at peak recovery. Infectious virus was recovered from the PTFE filter and NIOSH cyclone samplers on the peak day of viral RNA recovery. Conclusion: The PTFE filter and NIOSH cyclone sampler are useful for influenza virus RNA and infectious virus collection and may be considered for clinical and environmental settings. K E Y W O R D S bioaerosol samplers, bioaerosols, ferret model, influenza virus | 565 BEKKING Et al.

The transmission of infectious microbes via bioaerosols is of significant concern for both human and animal health. However, gaps in our understanding of respiratory pathogen transmission and methodological heterogeneity persist. New developments have enabled progress in this domain, and one of the major turning points has been the recognition that cross-disciplinary collaborations across spheres of human and animal health, microbiology, biophysics, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. Collaborative initiatives support advances in topics such as bioaerosol behavior, dispersion models, risk assessment, risk/exposure effects, and mitigation strategies in clinical, experimental, agricultural, and other field settings. There is a need to enhance the knowledge translation for researchers, stakeholders, and private partners to support a growing network of individuals and agencies to achieve common goals to mitigate inter- and intra-species pathogen transmission via bioaerosols.

The aerosolization mechanisms of viruses are poorly known, because of the challenges related to their sampling and observation. For example, single particle studies are needed to improve our understanding of bioaerosol enrichment processes. Such studies would help to develop models of airborne disease propagation. We propose a novel approach to study viral aerosols in single particles using a combination of fluorescence and transmission electron microscopy (TEM). This method allows for rapid analysis of labeled bacteriophages aerosolized and captured on a black membrane filter. It also requires performing image analyses on fluorescent spots. TEM is necessary to determine a single bacteriophage dimensions. Thus, the clustering of bacteriophage PP01 in a single aerosol particle was investigated and found to give a comparable number of virions to what was observed with TEM. The impact of the GFP (green fluorescent protein) in the head of PP01 virion compared to wild type (WT) PP01 was also tested by comparing the clustering of similar bioaerosol sizes generated by the aerosolization of PP01 WT, PP01-GFP, and PP01-GFP labeled with syto-red dye. Surprisingly, the PP01 WT bioaerosols were enriched compared to the PP01-GFP ones (64.9 § 17.5% more). PP01-GFPs were also found to be more numerous compared to those produced by PP01-GFP labeled with syto-red dye (28.9 § 16.9% more). The aerosolization process might be dependent on the electrochemical properties of the viruses and the environment. Changes of this nature could affect the mechanism of the aerosol formation in natural forming aerosols as demonstrated in this study for artificially generated aerosols.