Characteristics of compounds with strong or weak nitrification inhibition in sewage.

To clarify the characteristics of compounds with strong or weak nitrification inhibition in sewage, 64 organic compounds including compounds registered in Pollutant Release and Transfer Register (PRTR) were evaluated in terms of their chemical structures and molecular weights. Nineteen compounds showed strong nitrification inhibition by testing with Nitrosomonas europaea. Compounds with thioamide structures had the lowest median value of EC50 (0.017 mg/L), followed by those with alkyne structures (0.121 mg/L), chlorophenol structures (0.300 mg/L), and then azole structures (0.365 mg/L). In contrast, 33 of the 64 compounds showed weak nitrification inhibition at a concentration of 10 mg/L, 27 of which were categorized into three main groups: long-chain alcohol structures, alkyne structures with a phenyl group, and aromatic structures. Most compounds with strong nitrification inhibition had a low molecular weight (MW) from 50 to 200. Meanwhile, the proportion of compounds with weak nitrification inhibition tended to be greater with increasing MW and such compounds were predominant at higher molecular weights above 300. The correlations of results derived from tests of nitrification inhibition based on ISO 9509 and N. europaea showed that 24 out of 30 compounds provided results that were highly correlated between these tests (R = 0.85), while 4 compounds with chlorophenol structures and 2 compounds with alkyne structures showed weaker inhibition rates in the ISO 9509 test than in the N. europaea test. Our results indicate that the magnitude of nitrification inhibition depends on MW in addition to the chemical structure, which is helpful in the search for the cause of nitrification inhibition in wastewater treatment plants.

Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis.

Burkholderia stabilis strain FERMP-21014 secretes cholesterol esterase (BsChe), which is used in clinical settings to determine serum cholesterol levels. Previously, we constructed an expression plasmid with an endogenous constitutive promoter to enable the production of recombinant BsChe. In this study, we obtained one mutant strain with 13.1-fold higher BsChe activity than the wild type, using N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen. DNA-sequencing analysis revealed that the strain had lost chromosome 3 (∆Chr3), suggesting that the genes hindering BsChe production may be encoded on Chr3. We also identified common mutations in the functionally unknown BSFP_068720/30 genes in the top 10 active strains generated during transposon mutagenesis. As BSFP_068720/30/40 comprised an operon on Chr3, we created the BSFP_068720/30/40 disruption mutant and confirmed that each disruption mutant containing the expression plasmid exhibited ~ 16.1-fold higher BsChe activity than the wild type. Quantitative PCR showed that each disruption mutant and ΔChr3 had a ~ 9.4-fold higher plasmid copy number than the wild type. Structural prediction models indicate that BSFP_068730/40 is structurally homologous to the structural maintenance of chromosomes (SMC) protein MukBE, which is responsible for chromosome segregation during cell division. Conversely, BSFP_068720/30/40 disruption did not lead to a Chr3 drop-out. These results imply that BSFP_068720/30/40 is not a SMC protein but is involved in destabilizing foreign plasmids to prevent the influx of genetic information from the environment. In conclusion, the disruption of BSFP_068720/30/40 improved plasmid stability and copy number, resulting in exceptionally high BsChe production. KEY POINTS: • Disruption of BSFP_068720/30/40 enabled mass production of Burkholderia Che/Lip. • BSFP_068730/40 is an SMC protein homolog not involved in chromosome retention. • BSFP_068720/30/40 is likely responsible for the exclusion of exogenous plasmids.

Freshwater alga Raphidocelis subcapitata undergoes metabolomic changes in response to electrostatic adhesion by micrometer-sized nylon 6 particles.

Nylon powders are a type of microplastic (MP) used in personal care products such as cosmetics and sunscreens. To determine the effects of nylon polymers on freshwater microalgae, we investigated the effects of two types of micrometer-sized nylon polymers, i.e., powdered nylon 6 (Ny6-P) and nylon 12 (Ny12), and four other micrometer-sized MPs, i.e., low-density polyethylene, polyethylene terephthalate, polystyrene, and ultra-high-molecular-weight polyethylene, on the microalga Raphidocelis subcapitata. The results showed that Ny6-P inhibited R. subcapitata growth more than the other MPs; R. subcapitata growth was inhibited by 54.2% with 6.25 mg/L Ny6-P compared with the control. Ny6-P in the culture media adhered to R. subcapitata cells electrostatically, which may have disrupted growth and photosynthetic activity. Metabolomic analysis revealed that many metabolites related to the amino acid catabolic pathway and γ-glutamyl cycle were induced, which might trigger responses to avoid starvation and oxidative stress. Our study provides important information on the effects of Ny6-P on algae in freshwater environments.

Production of recombinant extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis.

Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL-1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.

Effect of lower chlorinated hydroxylated-polychlorobiphenyls on development of PC12 cells.

Hydroxylated polychlorobiphenyls (OH-PCBs) are major metabolites of PCBs that are widely distributed in the environment. While the effects of penta- to hepta-chlorinated OH-PCBs on neuronal differentiation have been widely reported, those of lower chlorinated OH-PCBs have not been extensively studied. To investigate the effects of lower chlorinated OH-PCBs on neuronal development, we studied the effects of mono- to hexa-chlorinated OH-PCBs on PC12 cells. Morphological changes were examined using an automatic system IN Cell Analyzer. Seventeen of the 20 OH-PCBs investigated promoted neuronal elongation in an OH-PCB concentration-dependent manner, while three OH-PCB congeners suppressed neuronal elongation based on Dunnett's analysis. In particular, the top five OH-PCBs (4OH-PCB2, 4'OH-PCB3, 4'OH-PCB25, 4'OH-PCB68, and 4'OH-PCB159), which have hydroxyl groups at the para-position and chlorine substitutions at the 2, 4, or 3' positions, significantly promoted neuronal elongation. Moreover, these neuronal elongations were suppressed by U0126, and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was observed in PC12 cells treated with 4OH-PCB2, 4'OH-PCB25, and 4'OH-PCB159. Taken together, our results indicate that the effect of OH-PCB on neuronal development is not dependent on the number of chlorine groups but on the chemical structure, and the mitogen-activated kinase kinase (MEK)-ERK1/2 signaling pathway is involved in this process.

Collection of DICOM RDSR (Digital Imaging and Communication in Medicine, Radiation Dose Structured Report) Information Aimed at Reducing Patient Exposure Dose.

The recent progress in angiography technology bestows benefits on patients for minimally invasive than surgery, while there has been an increase in the number of cases involving stochastic effects, such as radiation dermatitis, resulting from upgrading of the procedure because of an extension of the time for fluoroscopy and the number of shots. Recent CT equipment saves the dose data along with image data about the information management for patient exposure dose, which is used for management of individual cumulative dose and the presumed effective dose, using digital imaging and communication in medicine (DICOM). We extracted detailed information about shooting conditions and dose from the DICOM radiation dose structured report (DICOM RDSR) in the angiography area, and evaluated the trend of patient exposure dose in each procedure. As a result, we found that cases exceeding 3 Gy which needed observation in the head region were 16.7% and in the heart region were 27.3%. We also found that angiography had a higher dose of shooting than did fluoroscopy, and that the diagnosis and treatment with tumor involvement required a exposure dose than did vascular lesion. In this paper, we review the shooting conditions as a root of DICOM RDSR information and consider the possibility of planning for further reduction of the exposure dose.

Detoxification of hydroxylated polychlorobiphenyls by Sphingomonas sp. strain N-9 isolated from forest soil.

To examine the biodegradation of hydroxylated polychlorobiphenyls (OH-PCBs), we isolated Sphingomonas sp. strain N-9 from forest soil using mineral salt medium containing 4-hydroxy-3-chlorobiphenyl (4OH-3CB) at the concentration of 10 mg/L. Following incubation with strain N-9, the concentration of 4OH-3CB decreased in inverse proportion to strain N-9 proliferation, and it was converted to 3-chloro-4-hydroxybenzoic acid (4OH-3CBA) after 1 day. We observed that strain N-9 efficiently degraded lowly chlorinated OH-PCBs (1-4 Cl), while highly chlorinated OH-PCBs (5-6 Cl) were less efficiently transformed. Additionally, strain N-9 degraded PCBs and OH-PCBs with similar efficiencies, and the efficiency of OH-PCB degradation was dependent upon the positional relationships between OH-PCB hydroxyl groups and chlorinated rings. OH-PCB biodegradation may result in highly toxic products, therefore, we evaluated the cytotoxicity of two OH-PCBs [4OH-3CB and 4-hydroxy-3,5-dichlorobiphenyl (4OH-3,5CB)] and their metabolites [4OH-3CBA and 3,5-chloro-4-hydroxybenzoic acid (4OH-3,5CBA)] using PC12 rat pheochromocytoma cells. Our results revealed that both OH-PCBs induced cell membrane damage and caused neuron-like elongations in a dose-dependent manner, while similar results were not observed for their metabolites. These results indicated that strain N-9 can convert OH-PCBs into chloro-hydroxybenzoic acids having lower toxicity.

17ß-Trenbolone exposure programs metabolic dysfunction in larval medaka.

Here, we used physiological and transcriptomic analyses to evaluate the effects of 17ß-trenbolone (TB) on metabolism during the early life stage of medaka (Oryzias latipes). In the physiological experiments, sex reversal rates increased continuously in proportion to TB concentrations (2-100 ng/L), and were 100% (all males) in the 200 ng/L treatment group. TB caused a significant increase in the gonadosomatic index of females at concentrations of 60 and 100 ng/L. These females exhibited swollen abdomens and decreased egg production and fertility. Significant increases were observed in the body mass index of these females. TB caused decreased fertility in males at concentrations >20 ng/L, but no other effects were observed. In the transcriptomic (microarray) experiments, larvae were exposed to TB for up to 7 d. Analyses using the KEGG Orthology Database revealed that predominant categories of significantly upregulated genes included "lipid metabolism" and "metabolism of terpenoids and polyketides." Thirteen genes (including those for hydroxymethylglutaryl-CoA synthase, cytoplasmic synthase, and lanosterol synthase) related to cholesterol biosynthesis via the mevalonate pathway were highlighted in these categories. Reverse transcriptase-polymerase chain reaction analyses were consistent with the microarray results, in terms of the direction and magnitude of change to gene expression. Among the downregulated genes, angiopoietin-like 4 and mitochondrial uncoupling protein 1, which are inversely correlated with obesity, were detected in the TB treatments. In conclusion, the results suggest that the exposure of females to TB during the early life stage may cause metabolic dysfunctions, including obesity and disrupted cholesterol synthesis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1539-1551, 2016.

Genome-wide expression changes in Saccharomyces cerevisiae in response to high-LET ionizing radiation.

To understand the yeast response to high-linear energy transfer (LET) ionizing radiation (IR), we investigated global gene expression in yeast irradiated by three types of high-LET IR (fast neutrons, heavy ions, and thermal neutrons) and gamma rays using DNA microarray analysis. Stationary cells were irradiated by each IR and recultured in yeast-peptone-dextrose medium to allow repair for 40 min. RNA was then isolated from three independent samples of irradiated yeast. Genes involved in the Mec1p kinase pathway, which functions in DNA damage response, were induced by all forms of high-LET IR and by gamma rays. Some genes related to oxidative stress and the cell wall were induced by all forms of high-LET IRs. Gene expression patterns as a function of each type of high-LET IR were examined statistically by one-way analysis of variance. This analysis demonstrated the existence of irradiation-specific responses. For example, genes involved in ribosomal DNA synthesis were specifically induced by fast neutron irradiation, while the ubiquitin-proteasome system and heat shock response were specifically induced by thermal neutron irradiation. The study characterizes high-LET IR-induced gene expression and provides a molecular understanding of subsequent adaptation in yeast.

DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae.

Zinc pyrithione has been used in anti-dandruff shampoos and in anti-fouling paint on ships. However, little is known of its mode of action. We characterized the effects of sub-lethal concentrations of zinc pyrithione (Zpt) on Saccharomyces cerevisiae using DNA microarrays. The majority of the strongly upregulated genes are related to iron transport, and many of the strongly downregulated genes are related to the biosynthesis of cytochrome (heme). These data suggest that Zpt induces severe iron starvation. To confirm the DNA microarray data, we supplemented cultures containing Zpt with iron, and the growth of the yeast was restored significantly. From these results, we propose that the principal toxicity of zinc pyrithione arises from iron starvation.

Gene expression profiles in the brain of the neonate mouse perinatally exposed to methylmercury and/or polychlorinated biphenyls.

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are environmentally persistent neurodevelopmental toxicants. The primary source of human exposure is the consumption of contaminated fish, seafood and marine mammals. However, little is known about the molecular mechanisms of MeHg and PCB toxicities and interactions between these contaminants. We investigated the functional profiles of differently expressed genes in the brains of offspring mice perinatally exposed to MeHg and/or PCBs to elucidate how these contaminants interact with each other. Pregnant mice (C57BL/6) were divided into four groups by exposure: (1) vehicle control, (2) MeHg alone, (3) PCBs alone, (4) MeHg + PCBs. Gene expression analysis of the brains of offspring mice was carried out with 4 x 44 K whole mouse genome's microarrays (Agilent) on postnatal day 1. The gene expression pattern of the MeHg exposure-group differed from that of the PCB-exposure group. The MeHg + PCB group expressed a larger number of genes, most of which were not expressed in the MeHg group or PCB group. It was revealed that gene expression was greatly increased, and the most altered genes were found with co-exposure. The genes were related to the functional categories of development, inflammation, calcium ion homeostasis, signal transduction, the ubiquitin-proteasome pathway and detoxication. The ubiquitin-proteasome system and detoxication categories might function for protection against the toxicity induced by co-exposure to MeHg and PCBs. These results suggest that co-exposure does not simply exacerbate the toxicity of MeHg alone or PCB alone, but stimulates a protection system.

Toxicity of methanol and formaldehyde towards Saccharomyces cerevisiae as assessed by DNA microarray analysis.

To assess the toxicity of the C1 compounds methanol and formaldehyde, gene expression profiles of treated baker's yeast were analyzed using DNA microarrays. Among approximately 6,000 open reading frames (ORFs), 314 were repressed and 375 were induced in response to methanol. The gene process category "energy" comprised the greatest number of induced genes while "protein synthesis" comprised the greatest number of repressed genes. Products of genes induced by methanol were mainly integral membrane proteins or were localized to the plasma membrane. A total of 622 and 610 ORFs were induced or repressed by formaldehyde, respectively. More than one-third of the genes found to be strongly repressed by formaldehyde belonged to the "protein synthesis" functional category. Conversely, genes in the subcategory of "nitrogen, sulfur, and selenium metabolism" within "metabolism" and in the category of "cell rescue, defense, and virulence" were up-regulated by exposure to formaldehyde. Our data suggest that membrane structure is a major target of methanol toxicity, while proteins were major targets of formaldehyde toxicity.

A route to a Keggin-type alpha-[(X(III)O4)Mo12O35(OH)]4- anion through an Anderson-type [X(III)(OH)6Mo6O18]3- anion: X = Ga.

From an aqueous Mo(VI)-Ga(III)-HCl system, a colourless complex was isolated as a K(+) salt, which consists of a hexaprotonated Anderson-type [Ga(OH)(6)Mo(6)O(18)](3-) anion. A yellow complex became kinetically stable by the presence of CH(3)CN at concentrations of 30-40% (v/v). The X-ray structural analysis revealed that the yellow (NPr(n)(4))(4)[(GaO(4))Mo(12)O(35)(OH)] crystal contains an alpha-Keggin structure and the oxygen atom at an edge-shared contact is protonated. The formation conditions of the Keggin complex were elucidated in relation to those of the Anderson complex by a combined (71)Ga NMR and voltammetric study. Evidence was obtained of a spontaneous conversion of [Ga(OH)(6)Mo(6)O(18)](3-) to [(GaO(4))Mo(12)O(35)(OH)](4-) in the Mo(VI)-Ga(III) system.

Changes in gene expression of commercial baker's yeast during an air-drying process that simulates dried yeast production.

Changes in the gene expression of commercial baker's yeast during an air-drying process, which simulated dried yeast production, were analyzed. K-means clustering suggested that the genes involved in protein folding were transiently up-regulated at early stages, and that the genes involved in fatty acid metabolism were continuously up-regulated.

Self-cloning baker's yeasts that accumulate proline enhance freeze tolerance in doughs.

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding gamma-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.

Mechanisms of copper toxicity in Saccharomyces cerevisiae determined by microarray analysis.

The effect of the heavy metal copper on the expression of a wide spectrum of genes was analyzed by using a DNA microarray. The gene expression profile of baker's yeast Saccharomyces cerevisiae grown in a medium containing a sublethal concentration of cupric sulfate was compared with that of yeast grown in a normal medium. Among approximately 6000 yeast ORFs, 143 ORFs were induced more than twofold to resist copper toxicity after exposure to copper. Copper metallothionein CUP1-1 and CUP1-2 were induced more than 20-fold. Some genes related to sulfur metabolism and oxidative stress response were also up-regulated. This DNA microarray analysis identified several molecular targets of copper toxicity.

Functional genomics of commercial baker's yeasts that have different abilities for sugar utilization and high-sucrose tolerance under different sugar conditions.

In the modern baking industry, high-sucrose-tolerant (HS) and maltose-utilizing (LS) yeast were developed using breeding techniques and are now used commercially. Sugar utilization and high-sucrose tolerance differ significantly between HS and LS yeasts. We analysed the gene expression profiles of HS and LS yeasts under different sucrose conditions in order to determine their basic physiology. Two-way hierarchical clustering was performed to obtain the overall patterns of gene expression. The clustering clearly showed that the gene expression patterns of LS yeast differed from those of HS yeast. Quality threshold clustering was used to identify the gene clusters containing upregulated genes (cluster 1) and downregulated genes (cluster 2) under high-sucrose conditions. Clusters 1 and 2 contained numerous genes involved in carbon and nitrogen metabolism, respectively. The expression level of the genes involved in the metabolism of glycerol and trehalose, which are known to be osmoprotectants, in LS yeast was higher than that in HS yeast under sucrose concentrations of 5-40%. No clear correlation was found between the expression level of the genes involved in the biosynthesis of the osmoprotectants and the intracellular contents of the osmoprotectants. The present gene expression data were compared with data previously reported in a comprehensive analysis of a gene deletion strain collection. Welch's t-test for this comparison showed that the relative growth rates of the deletion strains whose deletion occurred in genes belonging to cluster 1 were significantly higher than the average growth rates of all deletion strains.

Mechanisms of patulin toxicity under conditions that inhibit yeast growth.

Patulin, 4-hydroxy-4H-furo[3,2c]pyran-2(6H)-one, is one of the best characterized and most widely disseminated mycotoxins found in agricultural products. Nonetheless, the mechanisms by which patulin causes toxicity are not well understood. Thus, the cytotoxicity of patulin was characterized by analysis of the yeast transcriptome upon challenge with patulin. Interestingly, patulin-induced yeast gene expression profiles were found to be similar to gene expression patterns obtained after treatment with the antifungal agricultural chemicals thiuram, maneb, and zineb. Moreover, patulin treatment was found to activate protein degradation, especially proteasome activities, sulfur amino acid metabolism, and the defense system for oxidative stress. Damage to DNA by alkylation was also suggested, and this seemed to be repaired by recombinational and excision repair mechanisms. Furthermore, the results provide potential biomarker genes for the detection of patulin in agricultural products. The results suggest the possibility of applying the yeast transcriptome system for the evaluation of chemicals, especially for natural chemicals that are difficult to get by organic synthesis.