Tadashi Nittami

Effects of Fe3O4 Magnetic Nanoparticles on A549 Cells

Fe3O4 magnetic nanoparticles (MgNPs-Fe3O4) are widely used in medical applications, including magnetic resonance imaging, drug delivery, and in hyperthermia. However, the same properties that aid their utility in the clinic may potentially induce toxicity. Therefore, the purpose of this study was to investigate the cytotoxicity and genotoxicity of MgNPs-Fe3O4 in A549 human lung epithelial cells. MgNPs-Fe3O4 caused cell membrane damage, as assessed by the release of lactate dehydrogenase (LDH), only at a high concentration (100 μg/mL); a lower concentration (10 μg/mL) increased the production of reactive oxygen species, increased oxidative damage to DNA, and decreased the level of reduced glutathione. MgNPs-Fe3O4 caused a dose-dependent increase in the CD44+ fraction of A549 cells. MgNPs-Fe3O4 induced the expression of heme oxygenase-1 at a concentration of 1 μg/mL, and in a dose-dependent manner. Despite these effects, MgNPs-Fe3O4 had minimal effect on cell viability and elicited only a small increase in the number of cells undergoing apoptosis. Together, these data suggest that MgNPs-Fe3O4 exert little or no cytotoxicity until a high exposure level (100 μg/mL) is reached. This dissociation between elevated indices of cell damage and a small effect on cell viability warrants further study.

Filamentous Bacterium Eikelboom Type 0092 in Activated Sludge Plants in Australia Is a Member of the Phylum Chloroflexi

ABSTRACT Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-β-hydroxyalkanoates or polyphosphate.

Acceleration of Vascular Sprouting from Fabricated Perfusable Vascular-Like Structures

Fabrication of vascular networks is essential for engineering three-dimensional thick tissues and organs in the emerging fields of tissue engineering and regenerative medicine. In this study, we describe the fabrication of perfusable vascular-like structures by transferring endothelial cells using an electrochemical reaction as well as acceleration of subsequent endothelial sprouting by two stimuli: phorbol 12-myristate 13-acetate (PMA) and fluidic shear stress. The electrochemical transfer of cells was achieved using an oligopeptide that formed a dense molecular layer on a gold surface and was then electrochemically desorbed from the surface. Human umbilical vein endothelial cells (HUVECs), adhered to gold-coated needles (ϕ600 μm) via the oligopeptide, were transferred to collagen gel along with electrochemical desorption of the molecular layer, resulting in the formation of endothelial cell-lined vascular-like structures. In the following culture, the endothelial cells migrated into the collagen gel and formed branched luminal structures. However, this branching process was strikingly slow (>14 d) and the cell layers on the internal surfaces became disrupted in some regions. To address these issues, we examined the effects of the protein kinase C (PKC) activator, PMA, and shear stress generated by medium flow. Addition of PMA at an optimum concentration significantly accelerated migration, vascular network formation, and its stabilization. Exposure to shear stress reoriented the cells in the direction of the medium flow and further accelerated vascular network formation. Because of the synergistic effects, HUVECs began to sprout as early as 3 d of perfusion culture and neighboring vascular-like structures were bridged within 5 d. Although further investigations of vascular functions need to be performed, this approach may be an effective strategy for rapid fabrication of perfusable microvascular networks when engineering three-dimensional fully vascularized tissues and organs.

Rapid engineering of endothelial cell-lined vascular-like structures in in situ crosslinkable hydrogels

Fabrication of perfusable vascular networks in vitro is one of the most critical challenges in the advancement of tissue engineering. Because cells consume oxygen and nutrients during the fabrication process, a rapid fabrication approach is necessary to construct cell-dense vital tissues and organs, such as the liver. In this study, we propose a rapid molding process using an in situ crosslinkable hydrogel and electrochemical cell transfer for the fabrication of perfusable vascular structures. The in situ crosslinkable hydrogel was composed of hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified hyaluronic acid (HA-CHO). By simply mixing these two solutions, the gelation occurred in less than 20 s through the formation of a stable hydrazone bond. To rapidly transfer cells from a culture surface to the hydrogel, we utilized a zwitterionic oligopeptide, which forms a self-assembled molecular layer on a gold surface. Human umbilical vein endothelial cells adhering on a gold surface via the oligopeptide layer were transferred to the hydrogel within 5 min, along with electrochemical desorption of the oligopeptides. This approach was applicable to cylindrical needles 200-700 µm in diameter, resulting in the formation of perfusable microchannels where the internal surface was fully enveloped with the transferred endothelial cells. The entire fabrication process was completed within 10 min, including 20 s for the hydrogel crosslinking and 5 min for the electrochemical cell transfer. This rapid fabrication approach may provide a promising strategy to construct perfusable vasculatures in cell-dense tissue constructs and subsequently allow cells to organize complicated and fully vascularized tissues while preventing hypoxic cell injury.

Filamentous members of cluster III Defluviicoccus have the in situ phenotype expected of a glycogen-accumulating organism in activated sludge.

The in situ ecophysiology of alphaproteobacterial filamentous Cluster III Defluviicoccus present in enhanced biological phosphorus removal (EBPR)-activated sludge systems was evaluated using FISH-MAR and histochemical staining methods. These organisms, sharing the Nostocoida limicola morphotype, are known to be responsible for serious episodes of activated sludge bulking. The data presented here also demonstrate an ability to assimilate short-chain fatty acids and synthesize poly-β-hydroxyalkanoates (PHA) anaerobically, and then utilize this stored PHA under aerobic conditions, but with no corresponding synthesis of polyphosphate. These features are consistent with an in situ phenotype of glycogen-accumulating organisms (GAO), populations thought to lower the efficiency of EBPR systems by outcompeting polyphosphate-accumulating organisms (PAO) for substrates in their anaerobic feed phase. Survey data indicate that these GAO are as commonly seen as the known PAO in full-scale EBPR-activated sludge systems, which suggest that they might play important roles there, and therefore should not be viewed just as laboratory curiosities.

Engineering thick cell sheets by electrochemical desorption of oligopeptides on membrane substrates

We developed a gold-coated membrane substrate modified with an oligopeptide layer that can be used to grow and subsequently detach a thick cell sheet through an electrochemical reaction. The oligopeptide CCRRGDWLC was designed to contain a cell adhesive domain (RGD) in the center and cysteine residues at both terminals. Cysteine contains a thiol group that forms a gold–thiolate bond on a gold surface. Cells attached to gold-coated membrane substrates via the oligopeptide layer were readily and noninvasively detached by applying a negative electrical potential to cleave the gold–thiolate bond. Because of the effective oxygen supply, fibroblasts vigorously grew on the membrane substrate and the thickness of the cell sheets was ∼60 μm at 14 days of culture, which was 2.9-fold greater than that of cells grown on a conventional culture dish. The cell sheets were detached after 7 min of electrical potential application. Using this approach, five layers of cell sheets were stacked sequentially with thicknesses reaching >200 μm. This approach was also beneficial for rapidly and readily transplanting cell sheets. Grafted cell sheets secreted collagen and remained at the transplanted site for at least 2 months after transplantation. This simple electrochemical cell sheet engineering technology is a promising tool for tissue engineering and regenerative medicine applications.

Comparison of Polytetrafluoroethylene Flat-Sheet Membranes with Different Pore Sizes in Application to Submerged Membrane Bioreactor

This study focused on phase separation of activated sludge mixed liquor by flat-sheet membranes of polytetrafluoroethylene (PTFE). A 20 liter working volume lab-scale MBR incorporating immersed PTFE flat-sheet membrane modules with different pore sizes (0.3, 0.5 and 1.0 μm) was operated for 19 days treating a synthetic wastewater. The experiment was interrupted twice at days 5 and 13 when the modules were removed and cleaned physically and chemically in sequence. The pure water permeate flux of each membrane module was measured before and after each cleaning step to calculate membrane resistances. Results showed that fouling of membrane modules with 0.3 μm pore size was more rapid than other membrane modules with different pore sizes (0.5 and 1.0 μm). On the other hand, it was not clear whether fouling of the 0.5 μm membrane module was more severe than that of the 1.0 μm membrane module. This was partly because of the membrane condition after chemical cleaning, which seemed to determine the fouling of those modules over the next period. When irreversible resistance (Ri) i.e., differences in membrane resistance before use and after chemical cleaning was high, the transmembrane pressure increased quickly during the next period irrespective of membrane pore size.

Fluorescence in situ hybridization probes targeting members of the phylum Candidatus Saccharibacteria falsely target Eikelboom type 1851 filaments and other Chloroflexi members

The FISH probe TM7-305 is thought to target the filamentous Eikelboom morphotype 0041 as a member of the Candidatus ‘Saccharibacteria’ (formerly TM7) phylum. However, with activated sludge samples in both Japan and Australia, this probe hybridized consistently with filamentous bacteria fitting the description of the morphotype 1851, which also responded positively to the CHL1851 FISH probe designed to target Chloroflexi members of this morphotype. 16S rRNA clone libraries from samples containing type 1851 TM7-305-positive filaments yielded Chloroflexi clones with high sequence similarity to Kouleothrix aurantiaca. These contained a variant TM7-305 probe target site possessing weakly destabilizing mismatches insufficient to prevent probe hybridization. Furthermore, the TM7-905 FISH probe, designed to target members of the entire Candidatus ‘Saccharibacteria’ phylum, also hybridized with the filament morphotypes 0041/0675, which responded also to the phylum level Chloroflexi probes. Many Chloroflexi sequences have only a single base mismatch to the TM7-905 probe target sequence. When competitor probes for both the TM7-305 and TM7-905 Chloroflexi non-target sites were applied, no fluorescent signal was seen in any of the filamentous organisms also hybridizing with the aforementioned Chloroflexi probes. These data indicate that these competitor probes must be included in hybridizations when both the TM7-905 and TM7-305 FISH probes are applied, to minimize potential false positive FISH results.

Elongation pattern and fine structure of the sheaths formed by Thiothrix nivea and Thiothrix fructosivorans

Thiothrix strains are filamentous sulfur-oxidizing bacteria common in activated sludge. Some of the members, including Thiothrix nivea and T. fructosivorans, are known to form a microtubular sheath that covers a line of cells. The sheaths are assemblages of [→4)-β-d-GlcN-(1→4)-β-d-Glc-(1→]n modified with unusual deoxy sugars. In an attempt to elucidate the sheath-forming mechanism, the patterns of sheath formation and cell proliferation were determined in this study. Prior to analysis, both sheaths were confirmed to be highly de-N-acetylated. Sheaths in viable filaments were N-biotinylated followed by cultivation and then fluorescently immunostained. Epifluorescence microscopy of the filaments revealed ubiquitous elongation of the sheaths. For visualization of the cell proliferation pattern, the cell membrane was fluorescently stained. The epifluorescence images demonstrated that cell proliferation also proceeds ubiquitously, suggesting that sheath elongation proceeds surrounding an elongating cell. In addition, the fine structure of the Thiothrix filaments was analyzed by transmission electron microscopy employing a freeze-substitution technique. The micrographs of freeze-substituted filaments showed that the sheaths were thin and single layered. In contrast, the sheaths in chemically fixed filaments appeared thick and multilayered. Treatment with glutaraldehyde probably caused deformation of the sheaths. Supporting this possibility, the sheaths were found to be deformed or solubilized by N-acetylation.

Influence of the electron acceptor on nitrite reductase gene (nir) diversity in an activated sludge community.

Analyses of the nitrite reductase gene diversities (nirK and nirS) in an activated sludge community fed with both nitrite and glucose were conducted. Results suggest that the topology of nirK and nirS gene fragment-based phylogenetic trees is influenced more by the available electron acceptor than by the carbon source. A denitrification reactor was operated for 53 days and a clone library constructed when the denitrifying communities in the SBR were supplied with both nitrite and glucose. Half of the nirK and nearly all the nirS gene fragments formed a cluster that was separate from a cluster containing nirK and nirS sequences derived from other communities in nitrate-fed reactors. On the other hand, nirK and nirS fragments obtained with glucose as the carbon source were similar to those detected in communities fed with other carbon sources.