Harmful dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins in field samples and cell immunolocalization using antipalytoxin antibodies

Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.     

Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.     

Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1

A 4-nitrophenol (4-NP)-degrading bacterium was isolated from activated sludge and identified as a Rhodococcus sp. This bacterium, designated as strain PN1, could utilize 4-NP as a sole carbon, nitrogen and energy source. Degradation tests of 4-NP using cell suspensions of strain PN1 revealed that the degradation was induced by 4-NP and that 4-nitrocatechol (4-NC) was one of the metabolites. A gene library was constructed from the total DNA of strain PN1 and introduced into Rhodococcus rhodochrous ATCC 12674. Two recombinant strains showed 4-NP hydroxylase activity, and a 9.1-kb DNA fragment encoding the activity was isolated from one of the strains. In addition, a 2.4-kb smaller fragment expressing the activity was subcloned from the 9.1-kb fragment and sequenced. The sequence analysis showed that the fragment encodes a two-component 4-NP hydroxylase, the predicted amino acid sequence of which exhibits significant similarity to those of phenol hydroxylases and 4-hydroxyphenylacetate 3-hydroxylases belonging to the two-component flavin diffusible monooxygenase (TC-FDM) family proposed by Galán et al. (J. Bacteriol., 182, 627-636, 2000).     

Prognostic predictor with multiple fuzzy neural models using expression profiles from DNA microarray for metastases of breast cancer

Gene expression profiling data from DNA microarray were analyzed using the fuzzy neural network (FNN) modeling method for predicting the distant metastases of breast cancer. The best model consisting of five genes was able to predict metastases of breast cancer with 94% accuracy. Furthermore, 100% accuracy was achieved by majoritarian decision using only 25 genes from five noninferior models which were constructed independently. From the constructed model, gene expression rules, which may cause distant metastases, were explicitly extracted and 60% of the metastases cases could be explained by this rule. The FNN modeling method described in this paper enables precise extraction of significant biological markers affecting prognosis without prior knowledge.     

Dietary Casein and Soy Protein Isolate Modulate the Effects of Raffinose and Fructooligosaccharides on the Composition and Fermentation of Gut Microbiota in Rats

Although diet has an important influence on the composition of gut microbiota, the impact of dietary protein sources has only been studied to a minor extent. In this study, we examined the influence of different dietary protein sources regarding the effects of prebiotic oligosaccharides on the composition and metabolic activity of gut microbiota. Thirty female rats were fed casein and soy protein isolate with cellulose, raffinose (RAF), and fructooligosaccharides (FOS). Microbiota composition was examined by real‐time qPCR and denaturing gradient gel electrophoresis. Dietary protein source affected cecum microbiota; acetic acid concentration and Lactobacillus spp. populations were greater with soy protein than with casein. Prebiotic oligosaccharides had distinctive effects on gut microbiota; RAF increased the acetic acid concentration and Bifidobacterium spp. populations, and FOS increased the butyric acid concentration regardless of the dietary protein. Likewise, Bifidobacterium sp., Collinsella sp., and Lactobacillus sp. were detected in microbiota of the rats fed RAF, and Bacteroides sp., Roseburia sp., and Blautia sp. were seen in microbiota of the rats fed FOS. Interactions between dietary proteins and prebiotic oligosaccharides were observed with Clostridium perfringens group populations and cecum IgA concentration. RAF and FOS decreased C. perfringens group populations in casein‐fed rats, and the combination of soy protein and RAF substantially increased cecum IgA concentration. These results indicate that dietary proteins can differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, depending on the type of carbohydrate polymers involved.     

Spatial distributions and profiles of atmospheric polycyclic aromatic hydrocarbons in two industrial cities in Japan

The spatial distribution and concentration profiles of 39 vapor and particulate polycyclic aromatic hydrocarbons (PAHs) have been investigated in two Japanese industrial cities (Fuji and Shimizu; a summer and winter season in each). The concentrations of particulate PAHs in winter tended to be higher than those in the summer, but for vapor PAHs, this was not the case. Significant correlations (p < 0.01) were found between most of the PAH concentrations monitored in winter, suggesting the presence of common emission sources. To identify PAH spatial distributions and emission sources in the area, we created contour maps for PAHs monitored; this indicates that the distinctive local distributions correspond to the emission sources. PAH profiles based on benzo[e]pyrene (BeP) concentration, especially for certain relatively heavy molecular weight PAHs, showed differential behaviors among divided areas related to potential regional emission sources such as paper-making plants, power plants, and traffic. We conclude that the origins of atmospheric PAHs in the surveyed areas were dominated by not only traffic but also by stationary emission sources such as paper-making plants and power plants and that local distributions were dependent on the local wind direction.     

Comparison of Centralized and Decentralized Systems in Power System Restoration

This paper proposes a multiagent approach to decentralized power system restoration for distribution system networks and presents a comparison of centralized and decentralized systems for power system restoration. Numerous studies have been conducted on power system restoration problems. From the viewpoint of system structures, this research can be divided into two categories: centralized systems and decentralized systems. In this study, we solve a power system restoration problem for a power distribution network by two different methods: mathematical programming (MIP) and the proposed multiagent‐based method (MAS). The proposed multiagent system consists of several distributing substation agents (DSAGs) and load agents (LAGs). An LAG corresponds to the customer load, and a DSAG supplies electricity to the LAG. From the simulation results, it can be seen that the proposed system can reach the right solution by making use of only local information and that the solution quality is better than that of the centralized system (MIP). This means that the proposed multiagent restoration system is a promising approach to larger scale distribution networks.     

Osteocytes: Their Lacunocanalicular Structure and Mechanoresponses

Osteocytes connect with neighboring osteocytes and osteoblasts through their processes and form an osteocyte network. Shear stress on osteocytes, which is induced by fluid flow in the lacunae and canaliculi, has been proposed as an important mechanism for mechanoresponses. The lacunocanalicular structure is differentially developed in the compression and tension sides of femoral cortical bone and the compression side is more organized and has denser and thinner canaliculi. Mice with an impaired lacunocanalicular structure may be useful for evaluation of the relationship between lacunocanalicular structure and mechanoresponses, although their bone component cells are not normal. We show three examples of mice with an impaired lacunocanalicular structure. Ablation of osteocytes by diphtheria toxin caused massive osteocyte apoptosis, necrosis or secondary necrosis that occurred after apoptosis. Osteoblast-specific Bcl2 transgenic mice were found to have a reduced number of osteocyte processes and canaliculi, which caused massive osteocyte apoptosis and a completely interrupted lacunocanalicular network. Osteoblast-specific Sp7 transgenic mice were also revealed to have a reduced number of osteocyte processes and canaliculi, as well as an impaired, but functionally connected, lacunocanalicular network. Here, we show the phenotypes of these mice in physiological and unloaded conditions and deduce the relationship between lacunocanalicular structure and mechanoresponses.     

Measurement of liquid water content in cathode gas diffusion electrode of polymer electrolyte fuel cell

Water management in cathode gas diffusion electrode (GDE) of polymer electrolyte fuel cell (PEFC) is essential for high performance operation, because liquid water condensed in porous gas diffusion layer (GDL) and catalyst layer (CL) blocks oxygen transport to active reaction sites. In this study, the average liquid water content inside the cathode GDE of a low-temperature PEFC is experimentally and quantitatively estimated by the weight measurement, and the relationship between the water accumulation rate in the cathode GDE and the cell voltage is investigated. The liquid water behavior at the cathode is also visualized using an optical diagnostic, and the effects of operating conditions and GDL structures on the water transport in the cathode GDE are discussed. It is found that the liquid water content in the cathode GDE increases remarkably after starting the fuel cell operation due to the water production at the CL. At a high current density, the cell voltage drops suddenly after starting the operation in spite of a low water content in the cathode GDE. When the GDL thickness is increased, much water accumulates near the cathode CL and the fuel cell shuts down immediately after the operation. In the final section of this paper, the structure of cathode GDL that has several grooves for water removal is proposed to prevent water flooding and improve fuel cell performance. This groove structure is effective to promote the removal of the liquid water accumulated near the active catalyst sites.