Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

Engineering osteogenic microenvironments by combination of multilayers from collagen type I and chondroitin sulfate with novel cationic liposomes.

Cationic liposomes composed of a novel lipid (N-{6-amino-1-[N-(9Z) -octadec9-enylamino] -1-oxohexan-(2S) -2-yl} -N'- {2- [N, N-bis(2-aminoethyl) amino] ethyl} -2-hexadecylpropandiamide) (OO4) and dioleoylphosphatidylethanolamine (DOPE) possess high amounts of amino groups and are promising systems for lipofection. Moreover, these cationic liposomes can also be used as a polycationic entity in multilayer formation using layer-by-layer technique (LbL), which is a method to fabricate surface coatings by alternating adsorption of polyanions and polycations. Since liposomes are suitable for endocytosis by or fusion with cells, controlled release of their cargo on site is possible. Here, a polyelectrolyte multilayer (PEM) system was designed of chondroitin sulfate (CS) and collagen type I (Col I) by LbL technique with OO4/DOPE liposomes embedded in the terminal layers to create an osteogenic microenvironment. Both, the composition of PEM and cargo of the liposomes were used to promote osteogenic differentiation of C2C12 myoblasts as in vitro model. The internalization of cargo-loaded liposomes from the PEM into C2C12 cells was studied using lipophilic (Rhodamine-DOPE conjugate) and hydrophilic (Texas Red-labeled dextran) model compounds. Besides, the use of Col I and CS should mimic the extracellular matrix of bone for future applications such as bone replacement therapies. Physicochemical studies of PEM were done to characterize the layer growth, thickness, and topography. The adhesion of myoblast cells was also evaluated whereby the benefit of a cover layer of CS and finally Col I above the liposome layer was demonstrated. As proof of concept, OO4/DOPE liposomes were loaded with dexamethasone, a compound that can induce osteogenic differentiation. A successful induction of osteogenic differentiation of C2C12 cells with the novel designed liposome-loaded PEM system was shown. These findings indicate that designed OH4/DOPE loaded PEMs have a high potential to be used as drug delivery or transfection system for implant coating in the field of bone regeneration and other applications.

Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling.

Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

Fine Structural Analysis of Brefeldin A-Induced Compartment Formation After High-Pressure Freeze Fixation of Maize Root Epidermis: Compound Exocytosis Resembling Cell Plate Formation during Cytokinesis.

Formation of large perinuclear brefeldin A (BFA)-induced compartments is a characteristic feature of root apex cells, but it does not occur in shoot apex cells. BFA-induced compartments have been studied mostly using low resolution fluorescence microscopy techniques. Here, we have employed a high-resolution ultrastructural method based on ultra rapid freeze fixation of samples in order to study the formation of BFA-induced compartments in intact maize root epidermis cells in detail. This approach reveals five novel findings. Firstly, plant TGN/PGN elements are not tubular networks, as generally assumed, but rather vesicular compartments. Secondly, TGN/PGN vesicles interact with one another extensively via stalk-like connections and even fuse together via bridge-like structures. Thirdly, BFA-induced compartments are formed via extensive homotypic fusions of the TGN/PGN vesicles. Fourthly, multivesicular bodies (MVBs) are present within the BFA-induced compartments. Fifthly, mitochondria and small vacuoles accummulate abundantly around the large perinuclear BFA-induced compartments.

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots.

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H2O2 accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl3, intracellular accumulation of H2O2 was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H2O2 was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.