Bioluminescence Microscopy: Design and Applications

Bioluminescence imaging by microscopy is performed using an ultra-low-light imaging camera. Although imaging devices such as sensor and camera have been greatly improved over time, such improvements have not been attained commercially which are available for microscopes now. We previously optimized the optical system of a microscope for bioluminescence imaging using a short-focal-length imaging lens and evaluated this system with a conventional color charge-coupled device camera. Here, we describe the concept of bioluminescence microscope design using a short-focal-length imaging lens and some representative applications, including intracellular calcium imaging, imaging of clock gene promoter assays, and three-dimensional reconstruction of Drosophila larva. This system facilitates the acquisition of bioluminescence images of single live cells using luciferase, which is similar to fluorescence microscopy using a fluorescent protein

Ecophysiological Role and Function of Uncultured Chloroflexi in an Anammox Reactor

The coexistence of uncultured heterotrophic bacteria belonging to the phylum Chloroflexi has often been observed in anaerobic ammonium oxidation (anammox) reactors fed with synthetic nutrient medium without organic carbon compounds. To determine if coexisting Chloroflexi in anammox reactors scavenge organic matter derived from anammox bacterial cells, the present study was conducted to investigate the substrate uptake pattern of the uncultured Chloroflexi present in an anammox reactor and to clarify if they take up microbial products derived from anammox bacterial cells. To accomplish this, combined microautoradiography and fluorescence in situ hybridization (MAR-FISH) was conducted. Phylogenetic analysis revealed that 36% of the clones analyzed in this study were affiliated with Chloroflexi. The sequence similarities to Anaerolinea thermophila and Caldilinea aerophila within the phylum Chloroflexi were only 81.0-88.7% and 80.3-83.8%, respectively. The uncultured Chloroflexi were found to incorporate sucrose, glucose, and N-acetyl-glucosamine. The 14C-tracing experiment revealed that the uncultured Chloroflexi were clearly MAR-positive, indicating the utilization of decaying anammox bacterial cell materials. Taken together, these results indicate that coexisting uncultured Chloroflexi in anammox reactors scavenge organic compounds derived from anammox bacterial cells.This research was partially supported by an Assistance for Research and Development of Construction Technology grant from the Ministry of Land, Infrastructure, Transport and Tourism, and by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology

Biomass Yield Efficiency of the Marine Anammox Bacterium, “Candidatus Scalindua sp.,” is Affected by Salinity

The growth rate and biomass yield efficiency of anaerobic ammonium oxidation (anammox) bacteria are markedly lower than those of most other autotrophic bacteria. Among the anammox bacterial genera, the growth rate and biomass yield of the marine anammox bacterium “Candidatus Scalindua sp.” is still lower than those of other anammox bacteria enriched from freshwater environments. The activity and growth of marine anammox bacteria are generally considered to be affected by the presence of salinity and organic compounds. Therefore, in the present study, the effects of salinity and volatile fatty acids (VFAs) on the anammox activity, inorganic carbon uptake, and biomass yield efficiency of “Ca. Scalindua sp.” enriched from the marine sediments of Hiroshima Bay, Japan, were investigated in batch experiments. Differences in VFA concentrations (0–10 mM) were observed under varying salinities (0.5%–4%). Anammox activity was high at 0.5%–3.5% salinity, but was 30% lower at 4% salinity. In addition, carbon uptake was higher at 1.5%–3.5% salinity. The results of the present study clearly demonstrated that the biomass yield efficiency of the marine anammox bacterium “Ca. Scalindua sp.” was significantly affected by salinity. On the other hand, the presence of VFAs up to 10 mM did not affect anammox activity, carbon uptake, or biomass yield efficiency.This research was partially supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology by the Japan Society for the Promotion of Science (JSPS) Fellows (T. Awata)

PAHs contents in road dusts on principal roads collected nationwide in Japan and their influential factors

54 Road dust samples were collected in principal roads (n=37) and residential roads (n=17) nationwide in Japan from March 2010 to November 2012. Sixteen polycyclic aromatic hydrocarbons (PAHs) and ignition loss (IL) were determined. The total PAHs contents ranged from 62 to 6,325 ng g-1 with a geometric mean of 484 ng g-1. The IL ranged from 0.8-17% with a mean of 6%. The PAHs contents were correlated with the IL contents, and the IL contents were dependent on the population density. From the PAHs patterns analysis, the PAHs of road dust is considered to be majorly from the diesel emissions

A Rhabdomyosarcoma Arising in the Larynx of a Dog

A neoplastic nodular lesion, 2 × 3 cm in diameter, was found in the larynx of a 6-year-old spayed female dog. The tumor was ill-circumscribed, consisting histologically of large round cells with abundant cytoplasm interspersed with small round cells with less cytoplasm and occasional multinucleated cells (myotubes). Immunohistochemically, tumor cells were positive for myoglobin, desmin and vimentin in varying degrees, but negative for S-100 protein, GFAP or cytokeratin. Cytoplasmic myofilaments/myofibrils with a dense Z-line-like structure were seen, the fine structures of which were complemented by PTAH stain. Based on these findings, the tumor was diagnosed as a rhabdomyosarcoma, a very rare tumor in the larynx of dogs

Methanotrophic community composition based on pmoA genes in dissolved methane recovery and biological oxidation closed downflow hanging sponge reactors

Dissolved methane in the effluent of anaerobic wastewater treatment processes is unrecovered and released into the atmosphere as methane, a greenhouse gas. To prevent methane emissions from effluent, a post-treatment system consisting of two closed downflow hanging sponge (DHS) reactors for the recovery and biological oxidation of dissolved methane was developed. More than 99% of the dissolved methane was completely eliminated using this system under ambient temperatures for 1 year. In this study, the methanotrophic community composition of the two closed DHS reactors was investigated. The performance of the closed DHS reactor was evaluated at different heights of the reactor in summer and winter. The clone libraries and T-RFLP analyses based on the pmoA gene revealed that type I and type II methanotrophs were present in the closed DHS reactors. Furthermore, type I methanotrophs showed wide diversity and contained uncultured phylogenetic clusters of methanotrophs (FWs and LWs), while type II methanotrophs were dominated by Methylocystis- and Methylosarcina-related clusters. The relative abundance of type II methanotrophs increased during winter. The type I methanotroph population dynamically changed with height of the reactor. These results demonstrate the important role of methanotrophs in removal of dissolved methane from upflow anaerobic sludge blanket effluent treating sewage. © 2017 Elsevier B.V.Embargo Period 12 month

Stormwater inflow loading of PAHs into urban domestic wastewater treatment plant for separate sewer system

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants present in wastewater, and determination of their sources is important for their management in the environment. In this study, stormwater loading of PAHs during rainfall periods was evaluated for sewage inflow into a wastewater treatment plant for separate sewer system. To accomplish this, sewage inflow volumes, suspended solid concentrations, and PAH concentrations were measured during eight rainfall events and on two no-rainfall days at the inlet of the plant. Based on a comparison between the rainfall and no-rainfall loading quantified by the measurements, excess PAH loadings with stormwater were evaluated for the rainfall events. The relationship between rainfall intensity and stormwater loading was then used to evaluate long-term stormwater loadings of water and PAHs. Their contributions to the sewage inflow were 0.7 % and 1.0 % for one-year for water and the sum of 16 measured PAHs, respectively. Our measurements and estimates demonstrate that direct stormwater inflow is not a primary source of PAHs to the plant for this separate sewer system.This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Numbers 19760372

Nitrogen removal using an anammox membrane bioreactor at low temperature

Membrane bioreactors (MBRs) have the ability to completely retain biomass and are thus suitable for slowly growing anammox bacteria. In the present study, an anammox MBR was operated to investigate whether the anammox activity would remain stable at low temperature, without anammox biomass washout. The maximum nitrogen removal rates were 6.7 and 1.1 g-N L−1 day−1 at 35 and 15°C, respectively. Fluorescence in situ hybridization and 16S rRNA-based phylogenetic analysis revealed no change in the predominant anammox species with temperature because of the complete retention of anammox biomass in the MBR. These results indicate that the predominant anammox bacteria in the MBR cannot adapt to a low temperature during short-term operation. Conversely, anammox activity recovered rapidly after restoring the temperature from the lower value to the optimal temperature (35°C). The rapid recovery of anammox activity is a distinct advantage of using an MBR anammox reactor.This research was partially supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology by the Japan Society for the Promotion of Science (JSPS)

Influence of temperature and salinity on microbial structure of marine anammox bacteria

Anaerobic ammonium oxidation (anammox) is a type of biological oxidation mediated by a group of Planctomycete-like bacteria. Members of the genus “Candidatus Scalindua” are mainly found in marine environments, but not exclusively. This group is cultured using different inoculums and conditions; however, its optimal growth conditions are not clear. Additionally, little is known about the factors that influence on the activity and the selection of population of marine anammox bacteria. This study was conducted to investigate the influence of temperature and salinity on the marine anammox community. To accomplish this, an up-flow fixed-bed column reactor was operated, and quantitative FISH with probes specific to dominant marine anammox bacteria was conducted. Anammox activity was observed at 20 and 30°C, but not at 10°C. A nitrogen removal rate of 0.32 kg TN m-3 day-1 was obtained at 20°C. These results suggest that temperature affects the activity (nitrogen removal rate) of anammox bacteria, while salinity does not affect the activity in the marine anammox biofilm.This study was partially supported by an Assistance for Research and Development of Construction Technology grant from the Ministry of Land, Infrastructure, Transport and Tourism, a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology, research foundations from the Institute for Fermentation (IFO), Osaka, and research foundations from the Public Association for Construction Service in the Chugoku region

In Vivo

Purpose. To compare the morphology of two types of vortex keratopathy: amiodarone-induced keratopathy and the Fabry disease-associated keratopathy. Patients and Methods. Eight patients who were receiving oral amiodarone therapy and 3 patients with Fabry disease, a mother and her 2 daughters, were examined by slit-lamp biomicroscopy and in vivo confocal microscopy (IVCM) regularly. Results. Amiodarone-induced keratopathy developed in 7 of the 8 patients, and it was detected as early as 7 days by IVCM and 14 days by slit-lamp biomicroscopy. The in vivo confocal microscopic images showed a clustering of corneal epithelial cells with a highly reflective cytoplasm in both types of keratopathy. In the amiodarone-induced keratopathy, the highly reflective epithelial cells were first found at the center of the cornea and then spread to the periphery with increasing time on amiodarone. In Fabry disease, the highly reflective epithelial cells were consistently observed extending from the limbus to the central cornea. Conclusion. These findings suggest that the corneal epithelial cells most likely endocytose amiodarone from the tear film in the amiodarone-induced keratopathy. In Fabry disease, globotriaosylceramide deposits are taken up by the lysosomes of the limbal epithelial stem cells, and they differentiate and migrate to the center of the cornea to form the whorl pattern