Effect of TiO2 NPs on the growth,anatomic features and biochemistry parameters of Baby sun rose (Aptenia cordifolia)

Rapid commercialization, industrialization and the use of nanotechnology has led to an increase in the distribution of nanoparticles (NPs) in the environment. The most common metal oxide NPs which is present within products is Titanium dioxide (TiO₂). TiO₂ NPs have photocatalytic nature and can affect plant growth. The current study investigated the morphological, anatomical and biochemical features of Baby sun rose (Aptenia cordifolia) after exposure to different concentrations of TiO₂ nanoparticles (0, 1, 5, 10 and 20 mg L⁻¹). Treatment with TiO₂ NPs showed changes in the morphological features and increased photosynthetic pigmentation within the plant. An increase in the level of phenolics (12%) and flavonoid compounds (13%) was observed when plants were treated with moderate levels of TiO₂ NPs. A reduction in the diameter of the vascular bundles and increased thickening of the transverse wall were observed in several samples. The number of scattered vascular bundles in the stems increased. The morphological, biochemical, and anatomical responses of Baby sun rose indicates that plants can adapt to environments contaminated with up to 20 mg L⁻¹ TiO₂ NPs_. The cultivation of Baby sun rose plants in environments polluted with TiO₂ NPs is recommended. This study enhances the knowledge of the effect of TiO₂ NPs on the growth of Baby sun rose which is an ornamental plant, widely cultivated in different regions of Iran. The results of this study suggest that contaminated environments up to 20 mg L⁻¹ TiO₂ NPs can be managed by phytoremediation. Further studies are needed to investigate this plant''s tolerance strategies against stress caused by TiO₂ NPs and bulk TiO₂ as well as the effect of other nanoparticles on plant.     

Fate and Phytotoxicity of CeO2 Nanoparticles on Lettuce Cultured in the Potting Soil Environment

Cerium oxide nanoparticles (CeO₂ NPs) have been shown to have significant interactions in plants. Previous study reported the specific-species phytotoxicity of CeO₂ NPs by lettuce (Lactuca sativa), but their physiological impacts and vivo biotransformation are not yet well understood, especially in relative realistic environment. Butterhead lettuce were germinated and grown in potting soil for 30 days cultivation with treatments of 0, 50, 100, 1000 mg CeO₂ NPs per kg soil. Results showed that lettuce in 100 mg·kg^-1 treated groups grew significantly faster than others, but significantly increased nitrate content. The lower concentrations treatment had no impact on plant growth, compared with the control. However, the higher concentration treatment significantly deterred plant growth and biomass production. The stress response of lettuce plants, such as Superoxide dismutase (SOD), Peroxidase (POD), Malondialdehyde(MDA) activity was disrupted by 1000 mg·kg^-1 CeO₂ NPs treatment. In addition, the presence of Ce (III) in the roots of butterhead lettuce explained the reason of CeO₂ NPs phytotoxicity. These findings demonstrate CeO₂ NPs modification of nutritional quality, antioxidant defense system, the possible transfer into the food chain and biotransformation in vivo.     

Acute Toxicity,Respiratory Reaction,and Sensitivity of Three Cyprinid Fish Species Caused by Exposure to Four Heavy Metals

Using 3 cyprinid fish species zebra fish, rare minnow, and juvenile grass carp, we conducted assays of lethal reaction and ventilatory response to analyze sensitivity of the fish to 4 heavy metals. Our results showed that the 96 h LC₅₀ of Hg²⁺ to zebra fish, juvenile grass carp, and rare minnow were 0.14 mg L⁻¹, 0.23 mg L⁻¹, and 0.10 mg L⁻¹, respectively; of Cu²⁺0.17 mg L⁻¹, 0.09 mg L⁻¹, and 0.12 mg L⁻¹ respectively; of Cd²⁺6.5 mg L⁻¹, 18.47 mg L⁻¹, 5.36 mg L⁻¹, respectively; and of Zn²⁺44.48 mg L⁻¹, 31.37 mg L⁻¹, and 12.74 mg L⁻¹, respectively. Under a 1-h exposure, the ventilatory response to the different heavy metals varied. Ventilatory frequency (Vf) and amplitude (Va) increased in zebra fish, juvenile grass carp, and rare minnows exposed to Hg²⁺ and Cu²⁺ (P<0.05), and the Vf and Va of the 3 species rose initially and then declined when exposed to Cd²⁺. Zn²⁺ had markedly different toxic effects than the other heavy metals, whose Vf and Va gradually decreased with increasing exposure concentration (P<0.05). The rare minnow was the most highly susceptible of the 3 fish species to the heavy metals, with threshold effect concentrations (TEC) of 0.019 mg L⁻¹, 0.046 mg L⁻¹, 2.142 mg L⁻¹, and 0.633 mg L⁻¹ for Hg²⁺, Cu²⁺, Cd²⁺, and Zn²⁺, respectively. Therefore, it is feasible to use ventilatory parameters as a biomarker for evaluating the pollution toxicity of metals and to recognize early warning signs by using rare minnows as a sensor.     

Toxicity of TiO2 Nanoparticles to Escherichia coli: Effects of Particle Size,Crystal Phase and Water Chemistry

Controversial and inconsistent results on the eco-toxicity of TiO₂ nanoparticles (NPs) are commonly found in recorded studies and more experimental works are therefore warranted to elucidate the nanotoxicity and its underlying precise mechanisms. Toxicities of five types of TiO₂ NPs with different particle sizes (10∼50 nm) and crystal phases were investigated using Escherichia coli as a test organism. The effect of water chemistry on the nanotoxicity was also examined. The antibacterial effects of TiO₂ NPs as revealed by dose-effect experiments decreased with increasing particle size and rutile content of the TiO₂ NPs. More bacteria could survive at higher solution pH (5.0–10.0) and ionic strength (50–200 mg L⁻¹ NaCl) as affected by the anatase TiO₂ NPs. The TiO₂ NPs with anatase crystal structure and smaller particle size produced higher content of intracellular reactive oxygen species and malondialdehyde, in line with their greater antibacterial effect. Transmission electron microscopic observations showed the concentration buildup of the anatase TiO₂ NPs especially those with smaller particle sizes on the cell surfaces, leading to membrane damage and internalization. These research results will shed new light on the understanding of ecological effects of TiO₂ NPs.     

Effects of Silver Nanoparticle Exposure on Germination and Early Growth of Eleven Wetland Plants

The increasing commercial production of engineered nanoparticles (ENPs) has led to concerns over the potential adverse impacts of these ENPs on biota in natural environments. Silver nanoparticles (AgNPs) are one of the most widely used ENPs and are expected to enter natural ecosystems. Here we examined the effects of AgNPs on germination and growth of eleven species of common wetland plants. We examined plant responses to AgNP exposure in simple pure culture experiments (direct exposure) and for seeds planted in homogenized field soils in a greenhouse experiment (soil exposure). We compared the effects of two AgNPs–20-nm polyvinylpyrrolidine-coated silver nanoparticles (PVP-AgNPs) and 6-nm gum arabic coated silver nanoparticles (GA-AgNPs)–to the effects of AgNO₃ exposure added at equivalent Ag concentrations (1, 10 or 40 mg Ag L⁻¹). In the direct exposure experiments, PVP-AgNP had no effect on germination while 40 mg Ag L⁻¹ GA-AgNP exposure significantly reduced the germination rate of three species and enhanced the germination rate of one species. In contrast, 40 mg Ag L⁻¹ AgNO₃ enhanced the germination rate of five species. In general root growth was much more affected by Ag exposure than was leaf growth. The magnitude of inhibition was always greater for GA-AgNPs than for AgNO₃ and PVP-AgNPs. In the soil exposure experiment, germination effects were less pronounced. The plant growth response differed by taxa with Lolium multiflorum growing more rapidly under both AgNO₃ and GA-AgNP exposures and all other taxa having significantly reduced growth under GA-AgNP exposure. AgNO₃ did not reduce the growth of any species while PVP-AgNPs significantly inhibited the growth of only one species. Our findings suggest important new avenues of research for understanding the fate and transport of NPs in natural media, the interactions between NPs and plants, and indirect and direct effects of NPs in mixed plant communities.     

Chlorophyll a Fluorescence and Photosynthetic and Growth Responses of Pinus radiata to Phosphorus Deficiency, Drought Stress, and High CO(2)

Needles from phosphorus deficient seedlings of Pinus radiata D. Don grown for 8 weeks at either 330 or 660 microliters CO₂ per liter displayed chlorophyll a fluorescence induction kinetics characteristic of structural changes within the thylakoid chloroplast membrane, i.e. constant yield fluorescence (F_O) was increased and induced fluorescence ([F_P-F_I]/F_O) was reduced. The effect was greatest in the undroughted plants grown at 660 μl CO₂ L⁻¹. By week 22 at 330 μl CO₂ L⁻¹ acclimation to P deficiency had occurred as shown by the similarity in the fluorescence characteristics and maximum rates of photosynthesis of the needles from the two P treatments. However, acclimation did not occur in the plants grown at 660 μl CO₂ L⁻¹. The light saturated rate of photosynthesis of needles with adequate P was higher at 660 μl CO₂ L⁻¹ than at 330 μl CO₂ L⁻¹, whereas photosynthesis of P deficient plants showed no increase when grown at the higher CO₂ concentration. The average growth increase due to CO₂ enrichment was 14% in P deficient plants and 32% when P was adequate. In drought stressed plants grown at 330 μl CO₂ L⁻¹, there was a reduction in the maximal rate of quenching of fluorescence (R_Q) after the major peak. Constant yield fluorescence was unaffected but induced fluorescence was lower. These results indicate that electron flow subsequent to photosystem II was affected by drought stress. At 660 μl CO₂ L⁻¹ this response was eliminated showing that CO₂ enrichment improved the ability of the seedlings to acclimate to drought stress. The average growth increase with CO₂ enrichment was 37% in drought stressed plants and 19% in unstressed plants.     

Metabolic Engineering of Rational Screened Saccharopolyspora spinosa for the Enhancement of Spinosyns A and D Production

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-O-methylated rhamnose which are derived from S-adenosylmethionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was 244 mg L⁻¹ and 129 mg L⁻¹, which was 4.88-fold and 4.77-fold higher than that in the wild-type (50 mg L⁻¹ and 27 mg L⁻¹), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong ermE* promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Herewith, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to 372/217 mg L⁻¹ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.     

Intracellular Conversion of Environmental Nitrate and Nitrite to Nitric Oxide with Resulting Developmental Toxicity to the Crustacean Daphnia magna

Background

Nitrate and nitrite (jointly referred to herein as NO_x) are ubiquitous environmental contaminants to which aquatic organisms are at particularly high risk of exposure. We tested the hypothesis that NO_x undergo intracellular conversion to the potent signaling molecule nitric oxide resulting in the disruption of endocrine-regulated processes.

Methodology/Principal Findings

These experiments were performed with insect cells (Drosophila S2) and whole organisms Daphnia magna. We first evaluated the ability of cells to convert nitrate (NO₃ ⁻) and nitrite (NO₂ ⁻) to nitric oxide using amperometric real-time nitric oxide detection. Both NO₃ ⁻ and NO₂ ⁻ were converted to nitric oxide in a substrate concentration-dependent manner. Further, nitric oxide trapping and fluorescent visualization studies revealed that perinatal daphnids readily convert NO₂ ⁻ to nitric oxide. Next, daphnids were continuously exposed to concentrations of the nitric oxide-donor sodium nitroprusside (positive control) and to concentrations of NO₃ ⁻ and NO₂ ⁻. All three compounds interfered with normal embryo development and reduced daphnid fecundity. Developmental abnormalities were characteristic of those elicited by compounds that interfere with ecdysteroid signaling. However, no compelling evidence was generated to indicate that nitric oxide reduced ecdysteroid titers.

Conclusions/Significance

Results demonstrate that nitrite elicits developmental and reproductive toxicity at environmentally relevant concentrations due likely to its intracellular conversion to nitric oxide.     

Thermotolerance of Leaf Discs from Four Isoprene-Emitting Species Is Not Enhanced by Exposure to Exogenous Isoprene

The effects of exogenously supplied isoprene on chlorophyll fluorescence characteristics were examined in leaf discs of four isoprene-emitting plant species, kudzu (Pueraria lobata [Willd.] Ohwi.), velvet bean (Mucuna sp.), quaking aspen (Populus tremuloides Michx.), and pussy willow (Salix discolor Muhl). Isoprene, supplied to the leaves at either 18 μL L⁻¹ in compressed air or 21 μL L⁻¹ in N₂, had no effect on the temperature at which minimal fluorescence exhibited an upward inflection during controlled increases in leaf-disc temperature. During exposure to 1008 μmol photons m⁻² s⁻¹ in an N₂ atmosphere, 21 μL L⁻¹ isoprene had no effect on the thermally induced inflection of steady-state fluorescence. The maximum quantum efficiency of photosystem II photochemistry decreased sharply as leaf-disc temperature was increased; however, this decrease was unaffected by exposure of leaf discs to 21 μL L⁻¹ isoprene. Therefore, there were no discernible effects of isoprene on the occurrence of symptoms of high-temperature damage to thylakoid membranes. Our data do not support the hypothesis that isoprene enhances leaf thermotolerance.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Anilofos Tolerance and Its Mineralization by the Cyanobacterium Synechocystis sp. Strain PUPCCC 64

This study deals with anilofos tolerance and its mineralization by the common rice field cyanobacterium Synechocystis sp. strain PUPCCC 64. The organism tolerated anilofos up to 25 mg L⁻¹. The herbicide caused inhibitory effects on photosynthetic pigments of the test organism in a dose-dependent manner. The organism exhibited 60, 89, 96, 85 and 79% decrease in chlorophyll a, carotenoids, phycocyanin, allophycocyanin and phycoerythrin, respectively, in 20 mg L⁻¹ anilofos on day six. Activities of superoxide dismutase, catalase and peroxidase increased by 1.04 to 1.80 times over control cultures in presence of 20 mg L⁻¹ anilofos. Glutathione content decreased by 26% while proline content was unaffected by 20 mg L⁻¹ anilofos. The test organism showed intracellular uptake and metabolized the herbicide. Uptake of herbicide by test organism was fast during initial six hours followed by slow uptake until 120 hours. The organism exhibited maximum anilofos removal at 100 mg protein L⁻¹, pH 8.0 and 30°C. Its growth in phosphate deficient basal medium in the presence of anilofos (2.5 mg L⁻¹) indicated that herbicide was used by the strain PUPCCC 64 as a source of phosphate.     

Partial Optimization of the 5-Terminal Codon Increased a Recombination Porcine Pancreatic Lipase (opPPL) Expression in Pichia pastoris

Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P.pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), K_m (0.041 mM), and V_max (2.008 µmol.mg protein ⁻¹.min⁻¹) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg.L⁻¹ vs 36 mg.L⁻¹) and total enzyme activity increased about 5 folds (1900 IU.L⁻¹ vs 367 IU.L⁻¹) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.     

Size-Dependent Antimicrobial Effects of Novel Palladium Nanoparticles

Investigating the interactions between nanoscale materials and microorganisms is crucial to provide a comprehensive, proactive understanding of nanomaterial toxicity and explore the potential for novel applications. It is well known that nanomaterial behavior is governed by the size and composition of the particles, though the effects of small differences in size toward biological cells have not been well investigated. Palladium nanoparticles (Pd NPs) have gained significant interest as catalysts for important carbon-carbon and carbon-heteroatom reactions and are increasingly used in the chemical industry, however, few other applications of Pd NPs have been investigated. In the present study, we examined the antimicrobial capacity of Pd NPs, which provides both an indication of their usefulness as target antimicrobial compounds, as well as their potency as potential environmental pollutants. We synthesized Pd NPs of three different well-constrained sizes, 2.0±0.1 nm, 2.5±0.2 nm and 3.1±0.2 nm. We examined the inhibitory effects of the Pd NPs and Pd²⁺ ions toward gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacterial cultures throughout a 24 hour period. Inhibitory growth effects of six concentrations of Pd NPs and Pd²⁺ ions (2.5×10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ M) were examined. Our results indicate that Pd NPs are generally much more inhibitory toward S. aureus than toward E. coli, though all sizes are toxic at ≥10⁻⁵ M to both organisms. We observed a significant difference in size-dependence of antimicrobial activity, which differed based on the microorganism tested. Our work shows that Pd NPs are highly antimicrobial, and that fine-scale (<1 nm) differences in size can alter antimicrobial activity.     

Phosphorus Accumulation and Sorption in Calcareous Soil under Long-Term Fertilization

Application of phosphorus (P) fertilizers to P-deficient soils can also result in P accumulation. In this study, soil P status and P uptake by apple trees were investigated in 5-, 10-, and 15-year-old orchards in the semi-arid Loess Plateau, China, and subset soils with different soil P statuses (14–90 Olsen-P mg kg⁻¹) were selected to evaluate the characteristic P adsorption. Due to the low P-use efficiency (4–6%), total soil P increased from 540 mg kg⁻¹ to 904 mg kg⁻¹, Olsen-P ranged from 3.4 mg kg⁻¹ to 30.7 mg kg⁻¹, and CaCl₂-P increased from less than 0.1 mg kg⁻¹ to 0.66 mg kg⁻¹ under continuous P fertilization. The P sorption isotherms for each apple orchard were found to fit the Langmuir isotherm model (R ² = 0.91–0.98). K (binding energy) and Q _m (P sorption maximum) decreased, whereas DPS (degree of phosphorus sorption) increased with increasing P concentration. CaCl₂-P increased significantly with the increase of Olsen-P, especially above the change point of 46.1 mg kg⁻¹. Application of surplus P could result in P enrichment in P-deficient soil which has high P fixation capacity, thus posing a significant environmental risk.     

The metabolic origins of non-photorespiratory CO2 release during photosynthesis: a metabolic flux analysis

Respiration in the light (R_L) releases CO₂ in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since R_L lowers net carbon fixation, understanding R_L could help improve plant carbon-use efficiency and models of crop photosynthesis. Although R_L was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to R_L, we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during ¹³CO₂ labeling time course, gas exchange, and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO₂ sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N₂; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that R_L releases 5.2 μmol CO₂ g⁻¹ FW h⁻¹ of CO₂, which is relatively consistent with a value of 9.3 μmol CO₂ g⁻¹ FW h⁻¹ measured by CO₂ gas exchange. The results indicated that ≤10% of R_L results from TCA cycle reactions, which are widely considered to dominate R_L. Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >93% of CO₂ released by R_L.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Effects of Irradiance during Growth on Adaptive Photosynthetic Characteristics of Velvetleaf and Cotton

We grew velvetleaf (Abutilon theophrasti Medic.) and cotton (Gossypium hirsutum L. var. Stoneville 213) at three irradiances and determined the photosynthetic responses of single leaves to a range of six irradiances from 90 to 2000 μeinsteins m⁻²sec⁻¹. In air containing 21% O₂, velvetleaf and cotton grown at 750 μeinsteins m⁻²sec⁻¹ had maximum photosynthetic rates of 18.4 and 21.9 mg of CO₂ dm⁻²hr⁻¹, respectively. Maximum rates for leaves grown at 320 and 90 μeinsteins m⁻²sec⁻¹ were 15.3 and 10.3 mg of CO₂ dm⁻²hr⁻¹ in velvetleaf and 12 and 6.7 mg of CO₂ dm⁻²hr⁻¹ in cotton, respectively. In 1 O₂, maximum photosynthetic rates were 1.5 to 2.3 times the rates in air containing 21% O₂, and plants grown at medium and high irradiance did not differ in rate. In both species, stomatal conductance was not significantly affected by growth irradiance. The differences in maximum photosynthetic rates were associated with differences in mesophyll conductance. Mesophyll conductance increased with growth irradiance and correlated positively with mesophyll thickness or volume per unit leaf area, chlorophyll content per unit area, and photosynthetic unit density per unit area. Thus, quantitative changes in the photosynthetic apparatus help account for photosynthetic adaptation to irradiance in both species. Net assimilation rates calculated for whole plants by mathematical growth analysis were closely correlated with single-leaf photosynthetic rates.     

Nitrogen, Carbon, and Sulfur Metabolism in Natural Thioploca Samples

Filamentous sulfur bacteria of the genus Thioploca occur as dense mats on the continental shelf off the coast of Chile and Peru. Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology. Thioploca is able to store internally high concentrations of sulfur globules and nitrate. It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate. We examined this nitrate reduction by incubation experiments of washed Thioploca sheaths with trichomes in combination with ¹⁵N compounds and mass spectrometry and found that these Thioploca samples produce ammonium at a rate of 1 nmol min⁻¹ mg of protein⁻¹. Controls showed no significant activity. Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min⁻¹ mg of protein⁻¹. The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate. The average sulfide oxidation rate measured was 5 nmol min⁻¹ mg of protein⁻¹ and could be increased to 10.7 nmol min⁻¹ mg of protein⁻¹ after the trichomes were starved for 45 h. Incorporation of ¹⁴CO₂ was at a rate of 0.4 to 0.8 nmol min⁻¹ mg of protein⁻¹, which is half the rate calculated from sulfide oxidation. [2-¹⁴C]acetate incorporation was 0.4 nmol min⁻¹ mg of protein⁻¹, which is equal to the CO₂ fixation rate, and no ¹⁴CO₂ production was detected. These results suggest that Thioploca species are facultative chemolithoautotrophs capable of mixotrophic growth. Microautoradiography confirmed that Thioploca cells assimilated the majority of the radiocarbon from [2-¹⁴C]acetate, with only a minor contribution by epibiontic bacteria present in the samples.     

Sunscreen Products as Emerging Pollutants to Coastal Waters

A growing awareness of the risks associated with skin exposure to ultraviolet (UV) radiation over the past decades has led to increased use of sunscreen cosmetic products leading the introduction of new chemical compounds in the marine environment. Although coastal tourism and recreation are the largest and most rapidly growing activities in the world, the evaluation of sunscreen as source of chemicals to the coastal marine system has not been addressed. Concentrations of chemical UV filters included in the formulation of sunscreens, such as benzophehone 3 (BZ-3), 4-methylbenzylidene camphor (4-MBC), TiO₂ and ZnO, are detected in nearshore waters with variable concentrations along the day and mainly concentrated in the surface microlayer (i.e. 53.6–577.5 ng L^-1 BZ-3; 51.4–113.4 ng L^-1 4-MBC; 6.9–37.6 µg L^-1 Ti; 1.0–3.3 µg L^-1 Zn). The presence of these compounds in seawater suggests relevant effects on phytoplankton. Indeed, we provide evidences of the negative effect of sunblocks on the growth of the commonly found marine diatom Chaetoceros gracilis (mean EC₅₀ = 125±71 mg L^-1). Dissolution of sunscreens in seawater also releases inorganic nutrients (N, P and Si forms) that can fuel algal growth. In particular, PO₄ ³⁻ is released by these products in notable amounts (up to 17 µmol PO₄ ³⁻ g⁻¹). We conservatively estimate an increase of up to 100% background PO₄ ³⁻ concentrations (0.12 µmol L^-1 over a background level of 0.06 µmol L^-1) in nearshore waters during low water renewal conditions in a populated beach in Majorca island. Our results show that sunscreen products are a significant source of organic and inorganic chemicals that reach the sea with potential ecological consequences on the coastal marine ecosystem.     

Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

Background

Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene hMLH1’s promoter is causal, the precise cause of EMAST is not clearly defined but points towards hMSH3 deficiency.

Aim

To examine if hMSH3 deficiency causes EMAST, and to explore mechanisms for its deficiency.

Methods

We measured −4 bp framshifts at D8S321 and D20S82 loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, hMLH1^−/−, hMSH6^−/−, and hMSH3^−/− CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.

Results

D8S321 mutations occurred 31-and 40-fold higher and D20S82 mutations occurred 82-and 49-fold higher in hMLH1^−/− and hMSH3^−/− cells, respectively, than in hMSH6^−/− or MMR-proficient cells. hMSH3 knockdown in MMR-proficient cells caused higher D8S321 mutation rates (18.14 and 11.14×10⁻⁴ mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻⁴ mutations/cell/generation; p<0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H₂O₂ to examine its effect on hMSH3. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H₂O₂ treatment.

Conclusion

EMAST is dependent upon the MMR background, with hMSH3^−/− more prone to frameshift mutations than hMSH6^−/−, opposite to frameshift mutations observed for mononucleotide repeats. hMSH3^−/− mimics complete MMR failure (hMLH1^−/−) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, hMSH3-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3’s subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.