Dynamic nutrient removal potential of a novel submerged macrophyte Rotala rotundifolia, and its growth and physiological response to reduced light available.

Rotala rotundifolia is a novel submerged macrophyte able to survive across the winter under temperature as low as 4 °C. Dynamic nutrient removal potential of R. rotundifolia was estimated using the Eco-tank system simulating natural eutrophic waters. The growth and physiological response of R. rotundifolia by cutting and division propagation to light (100%, 60%, and 20% natural light) were investigated. The results showed that R. rotundifolia was superior in removing N and P from eutrophic waters. As influent concentrations of NH4+-N and total phosphorus (TP) were 4.81-5.87 and 0.61-0.78 mg L-1, effluent concentrations of NH4+-N, total nitrogen (TN), and TP were separately 0.06-1.10, 0.40-1.59, and 0.05-0.17 mg L-1, with removal efficiencies of 93.6%, 84.6%, and 82.5% at a flow rate of 200 L d-1. The growth and morphology of the plant under two propagation patterns were influenced by light and the responses were quite different. The biomass of the plant by cutting was higher at low light conditions, and the plant allocated more biomass on above ground. However, there was no significant difference in the height. By division, the plant preferred to high light. The biomass and height were significantly higher at 100% natural light. The peroxidase (POD), superoxide dismutase (SOD) and root activities of plant by cutting showed a trend of decrease and followed by an increase with light reduction, while by division, they increased with reduced light available. Variations of chlorophyll and soluble protein of the plant by cutting and division were contrary to the changes of POD activity. These results suggest that R. rotundifolia can be used to effectively remove nitrogen and phosphorus in eutrophic waters, and high light promotes the growth of the plant by division, while suitable shade is needed for the plant by cutting.

Enhanced Production of Active Ecumicin Component with Higher Antituberculosis Activity by the Rare Actinomycete Nonomuraea sp. MJM5123 Using a Novel Promoter-Engineering Strategy.

Ecumicins are potent antituberculosis natural compounds produced by the rare actinomycete Nonomuraea sp. MJM5123. Here, we report an efficient genetic manipulation platform of this rare actinomycete. CRISPR/Cas9-based genome editing was achieved based on successful sporulation. Two genes in the ecumicin gene cluster were further investigated, ecuN and ecuE, which potentially encode a pretailoring cytochrome P450 hydroxylase and the core peptide synthase, respectively. Deletion of ecuN led to an enhanced ratio of the ecumicin compound EcuH16 relative to that of EcuH14, indicating that EcuN is indeed a P450 hydroxylase, and there is catalyzed hydroxylation at the C-3 position in unit12 phenylalanine to transform EcuH16 to the compound EcuH14. Furthermore, promoter engineering of ecuE by employing the strong promoter kasO*P was performed and optimized. We found that integrating the endogenous ribosome-binding site (RBS) of ecuE together with the RBS from kasO*P led to improved ecumicin production and resulted in a remarkably high EcuH16/EcuH14 ratio. Importantly, production of the more active component EcuH16 was considerably increased in the double RBSs engineered strain EPR1 compared to that in the wild-type strain, reaching 310 mg/L. At the same time, this production level was 2.3 times higher than that of the control strain EPA1 with only one RBS from kasO*P. To the best of our knowledge, this is the first report of genome editing and promoter engineering on the rare actinomycete Nonomuraea.

[Remediation performance and mechanism of aquatic plants for iron polluted water]. / 水生植物修复铁污染水体的效果及作用机制.

To solve the yellow colorization in water caused by iron ion, we evaluated the remediation performances of six aquatic plant species (Hygroryza aristata, Myriophyllum verticillatum, Hydrocotyle verticillata, Jussiaea stipulacea, Pistia stratiotes and Rotala rotundifolia) using hydroponic experiment. Effects of iron concentration, pH, plant biomass on iron removal were investigated, and the intensification of removing iron incurred by aeration was also discussed. Results showed that all the examined plant species could improve both divalent iron and total iron removal, but with significant difference in their performance. Divalent iron concentrations were decreased by H. aristata and H. verticillata from 5.0 mg·L-1 to 0.23 and 0.26 mg·L-1 within 24 h, respectively, meeting the standard of supplementary items for the drinking water and surface water sources (divalent iron concentration ≤0.3 mg·L-1), while total iron concentrations declined to 0.84 and 1.21 mg·L-1 with removal efficiency of 83.2% and 75.8%, respectively. Concentrations of divalent iron and total iron of plant treatment plots at pH 5, 6, 7, 8 were not significantly different, with removal efficiency of divalent iron and total iron being among 95.4%-98.4% and 92.2%-94.6%, separately. When initial divalent iron concentration was less than 5.0 mg·L-1, removal efficiency of divalent iron and total iron increased with the increases of divalent iron concentration. The growth of H. aristata was inhibited at divalent iron concentration of 10.0 mg·L-1. Total iron removal was not stable during the trial. Removal efficiency of plant treatment rose only by 7.0% compared with the control, which was much lower than other concentration treatments. The divalent iron concentration was decreased to ＜ 0.3 mg·L-1 in 24 h at plant biomass :300 g, with no difference of removal efficiency among biomass treatments. Both intermittent and continuous aeration enhanced iron removal by H. aristata, but continuous aeration was more favorable for the removal of total iron due to stabilization.

Nutrient removal by Rotala rotundifolia: a superior candidate for ecosystem remediation at low temperatures.

Temperature is an extremely important factor affecting the nutrient (mainly nitrogen and phosphorus) removal of aquatic macrophytes. A novel submersed Rotala rotundifolia was separately cultivated at room and low temperatures to investigate its ability for nutrient removal. The physiological metabolism was analyzed to explore the mechanism of removing nutrients under a wide temperature range. The results showed that the removal efficiency (RE) of nutrients at low temperature was competitive with that obtained at normal temperature, demonstrating that temperature exerted no obvious influence on the nutrient removal by R. rotundifolia. The root vitality at 5 °C rose from the initial 0.26 to 1.5 mg g-1 h-1, whereas it fell by 38.66% at 10 °C, 28.74% at 20 °C and 5.15% at 30 °C. The peroxidase (POD) activity at 5 °C showed the maximum value on day 7 followed by a notable decline on day 21. All the peak values of soluble sugar and protein as well as MDA showed up at 5 °C and they were 5.5, 437.9 and 10.1 mg g-1, respectively. Chlorophyll a and b reached 8.4 and 4.4 mg g-1 on day 28, respectively, with a total chlorophyll content (a plus b) of 12.4 mg g-1 at 5 °C, all of which were higher than that at 30 °C. These results validated that R. rotundifolia could be a superior candidate suitable for in situ application.

Energy footprint and carbon emission reduction using off-the-grid solar-powered mixing for lagoon treatment.

Mixing is the driver for the energy footprint of water resource recovery in lagoons. With the availability of solar-powered equipment, one potential measure to decrease the environmental impacts of treatment is to transition to an off-the-grid treatment. We studied the comparative scenarios of an existing grid-powered mixer and a solar-powered mixer. Testing was conducted to monitor the water quality, and to guarantee that the effluent concentrations were maintained equally between the two scenarios. Meanwhile, the energy consumption was recorded with the electrical energy monitor by the wastewater treatment utility, and the carbon emission changes were calculated using the emission intensity of the power utility. The results show that after the replacement, both energy usage and energy costs were significantly reduced, with the energy usage having decreased by 70% and its cost by 47%. Additionally, carbon-equivalent emission from electricity importation dropped by 64%, with an effect on the overall carbon emissions (i.e., including all other contributions from the process) decreasing from 3.8% to 1.5%.

Identification of Preferential Paths of Fossil Carbon within Water Resource Recovery Facilities via Radiocarbon Analysis.

The Intergovernmental Panel on Climate Change (IPCC) reported that all carbon dioxide (CO2) emissions generated by water resource recovery facilities (WRRFs) during treatment are modern, based on available literature. Therefore, such emissions were omitted from IPCC's greenhouse gas (GHG) accounting procedures. However, a fraction of wastewater's carbon is fossil in origin. We hypothesized that since the fossil carbon entering municipal WRRFs is mostly from soaps and detergents as dissolved organic matter, its fate can be selectively determined during the universally applied separation treatment processes. Analyzing radiocarbon at different treatment points within municipal WRRFs, we verified that the fossil content could amount to 28% in primary influent and showed varying distribution leaving different unit operations. We recorded the highest proportion of fossil carbon leaving the secondary treatment as off-gas and as solid sludge (averaged 2.08 kg fossil-CO2-emission-potential m-3 wastewater treated). By including fossil CO2, total GHG emission in municipal WRRFs increased 13%, and 23% if an on-site energy recovery system exists although much of the postdigestion fossil carbon remained in biosolids rather than in biogas, offering yet another carbon sequestration opportunity during biosolids handling. In comparison, fossil carbon contribution to GHG emission can span from negligible to substantial in different types of industrial WRRFs. With such a considerable impact, CO2 should be analyzed for each WRRF and not omitted from GHG accounting.

Silane-based coatings on the pyrite for remediation of acid mine drainage.

Acid mine drainage (AMD) resulting from the oxidation of pyrite and other metal sulfides has caused significant environmental problems, including acidification of rivers and streams as well as leaching of toxic metals. With the goal of controlling AMD at the source, we evaluated the potential of tetraethylorthosilicate (TEOS) and n-propyltrimethoxysilane (NPS) coatings to suppress pyrite oxidation. The release of total Fe and SO4(-2) from uncoated and coated pyrite in the presence of a chemical oxidizing agent (H2O2) or iron-oxidizing bacteria (Acidithiobacillus ferrooxidans) was measured. Results showed that TEOS- and NPS-based coatings reduced chemical oxidation of pyrite by as much as 59 and 96% (based on Fe release), respectively, while biological oxidation of pyrite was reduced by 69 and 95%, respectively. These results were attributed to the formation of a dense network of Fe-O-Si and Si-O-Si bonds on the pyrite surface that limited permeation of oxygen, water, and bacteria. Compared with results for TEOS-coated pyrite, higher pH and lower concentrations of total Fe and SO4(-2) were observed for oxidation of NPS-coated pyrite, which was attributed to its crack-free morphology and the presence of hydrophobic groups on the NPS-based coating surface. The silane-based NPS coating was shown to be highly effective in suppressing pyrite oxidation, making it a promising alternative for remediation of AMD at its source.

Gene expression microarray analysis of the effects of grape anthocyanins in mice: a test of a hypothesis-generating paradigm.

The mechanism(s) through which fruits, vegetables, and whole grains favorably affect health is not well established. Using an anthocyanin-rich grape as a model, we examined the ability of an agnostic analytical approach using gene expression microarrays to generate novel testable hypotheses regarding the mechanisms of action of potentially healthful foods and food components. C57BL/6 mice were divided into 2 groups and fed a proatherogenic diet with or without a semipurified anthocyanin extract (70% anthocyanins) incorporated at a level of 0.1 mg/mL into the drinking water. After 6 weeks, compared with control mice, mice supplemented with anthocyanins tended to gain more weight and have increased adipose tissue mass, although these effects did not achieve statistical significance. Anthocyanin-supplemented mice had significantly reduced relative liver weights and heart weights. Serum lipids and inflammatory cytokines were not different between the groups. Gene expression microarray analysis of the liver and skeletal muscle identified a number of molecular pathways significantly affected by anthocyanin treatment. Two distinct clusters emerged. The first cluster included down-regulated pathways in both muscle and liver involving cellular defense, whereas the second included hepatic genes involved in energy metabolism. From these data, 3 hypotheses were developed for future investigation.

Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions.

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.

Effect of N-acetylglucosaminyltransferase V on the expressions of other glycosyltransferases.

Transfection of sense cDNA of N-acetylglucosaminyltransferase V (GnTV) into H7721 human hepatocellular carcinoma cells resulted in the decreased expression of surface sialyl Lewis X (SLe(x)), a sialylated fucose-containing antigen. The enzymatic mechanisms were speculated to be the concomitantly decreased expression of alpha1,3-fucosyltransferase (FucT)-III, -VI, -VII and the branching enzyme of O-glycans, core 2-beta1,6-N-acetylglucosaminyltransferase (C2GnT)-I, -II. These two glycosyltransferase families were suggested to be the key enzymes in the synthesis of SLe(x). The expression of alpha2,3-sialyltransferase (ST3)-IV, but not ST3-I, -II and -III was elevated by sense GnTV. However, it did not cause the increase of SLe(x) synthesis. Transfection of antisense GnTV into H7721 cells showed entirely opposite effects on the expression of above-mentioned SLe(x) and glycosyltransferases as the sense GnTV.

Forskolin up-regulates metastasis-related phenotypes and molecules via protein kinase B, but not PI-3K, in H7721 human hepato-carcinoma cell line.

Forskolin (FSK) is known as an up-regulator of intracellular cAMP and inhibitor of cancer growth and metastasis. The effects of FSK on the metastasis potential and its mechanisms were studied using a human hepatocarcinoma cell line, H7721. It was found that FSK stimulated cell growth, increased cAMP in the cells, and enhanced the metastasis-related phenotypes, including adhesion to laminin (Ln) and human umbilical vein epithelial cells (HUVEC), chemotactic migration and invasion. These effects were supposed to result from the increase of the SLex expression induced by FSK, since only the monoclonal antibody of SLex showed a significant attenuation of the enhanced metastasis-associated phenotypes. Using H7721 cells transfected with the sense or antisense cDNA of protein kinase B (PKB) and some inhibitors of signal transduction, it was discovered that FSK up-regulated the expression of SLex via PKB, but it was independent of phosphotidylinositide-3-kinase (PI-3K). A subtype of atypical protein kinase C (a-PKC) might also participate in the up-regulation of SLex expression by FSK, and cAMP/PKA pathway is a negative regulator of SLex expression on H7721 cells. It can be concluded that FSK shows a metastasis-promoting effect ex vivo.

Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V.

The expressions of integrin alpha5, beta1, and alpha6 were studied in H7721 cells by means of flow cytometric and RT-PCR method after transfected with sense and antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V). The transfected cells were characterized by Northern blot. It was found that the order of expression from high to low was beta1>alpha5>alpha6. Transfection of sense GnT-V up-regulated alpha5 and alpha6, but not beta1 subunit, while antisense GnT-V down-regulated alpha5 and beta1, but not alpha6. The alterations of surface integrin subunits were quite compatible with the changes of their mRNAs. Using enzyme-labeled lectin analysis, it was shown that alpha5 subunit contained only C(2)C(2) biantennary N-glycan, which was not regulated by sense and antisense GnT-V. In contrast, beta1 subunit contained both biantennary and tri-/tetra-antennary N-glycans with GlcNAcbeta1,6Manalpha1,6-branch, and the latter was up- and down-regulated by the sense and antisense GnT-V, respectively. Therefore, the amount of biantennary N-glycans on beta1 subunit, but not the integrin protein, was correlated to the cell adhesion to fibronectin and laminin, which was reduced and elevated in the sense and antisense GnT-V-transfected cells, respectively, as we previously reported.

Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line.

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLe(x)), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLe(x) played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLe(x) and alpha-1,3-fucosyltransferase-VII (alpha-1,3 Fuc T-VII, enzyme for SLe(x) synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLe(x) and alpha-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLe(x) expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLe(x) and alpha-1,3 FucT-VII.

Alteration in the expression of early stage processing enzymes of N-glycan during myeloid and monocytoid differentiation of HL-60 cells.

The expressions of the enzymes participating in the early stage of N-glycan processing, Golgi alpha-Mase-I, alpha-Mase-II and GnT-I, GnT-II, were studied before and after HL-60 cells were differentiated to myelocytes or monocytes induced by ATRA or PMA, respectively. It was found that alpha-Mase-I activity and GnT-I mRNA were decreased by both ATRA and PMA, while alpha-Mase-II and GnT-II were altered insignificantly. The down-regulation of alpha-Mase-I and GnT-I was cell specific, since ATRA up-regulated alpha-Mase-I and GnT-I in the H7721 hepatocarcinoma cell line. However, in H7721 cells, PMA also decreased alpha-Mase-I and GnT-I, and both ATRA and PMA also did not obviously change the expressions of alpha-Mase-II and GnT-II.

Regulation of Phospholipase D Activity in Human Hepatocacinoma Cells by Protein Kinases and D-sphingosine.

The effects of some inhibitors of protein kinase C(PKC) and tyrosine protein kinase(TPK)as well as the antibodies to PKC isotypes on the activity of phosphatidylcholine-specific phospholipase D(PLD)in 7721 human hepatocarcinoma cells were determined in order to study the regulation of PKC and TPK on PLD in these cells. It was found that all of the four inhibitors of PKC (chelerythrine, H-7, calphostin C and stausporine) and two inhibitors of TPK (tyrphostin 46 and genistein) partially inhibited the basal activity of PLD. Among them, the inhibition rates of staurosporine and calphostin C were the highest. The effects of TPK inhibitors were less than that of PKC inhibitors. When the inhibitors of PKC and TPK were added in combination, the inhibitory effect was greater than that used separately. A well known physiological inhibitor of PKC, D-sphingosine, did not show any inhibition, but did show stimulation on PLD activity. The mechanism is probably related to the transformation of D-sphingosine to D-sphingosine 1-phosphate, a stimulator of PLD via the activation TPK (and probably also PKC). The stimulating effects of both D-sphingosine and D-sphingosine 1-phosphate were blocked by TPK inhibitors and other PKC inhibitors. Among the 3 common PKC isotypes in human hepatocarcinoma cells, PKCalpha and PKCbetaI may be the main isotypes of PKC in the regulation of PLD.