Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13C enriched biological samples using 1H-13C 2D heteronuclear correlation NMR spectroscopy

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, ¹H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and ¹H-¹³C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D ¹H-¹³C analysis of intact ¹³C enriched biological samples. Questions include the following: Can ¹H-¹³C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D ¹H-¹³C HSQC, HMQC, and HETCOR variants were compared and then applied to ¹³C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for ¹H-¹³C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.     

BLUE AND ULTRAVIOLET-B LIGHT PHOTORECEPTORS IN PARSLEY CELLS

Abstract— Ultraviolet-B (UV-B) and blue light photoreceptors have been shown to regulate chalcone synthase and flavonoid synthesis in parsley cell cultures. These photoreceptors have not yet been identified. In the present work, we studied UV-B photoreception with physiological experiments involving temperature shifts and examined the possible role of flavin in blue and UV-B light photoreception. Cells irradiated with UV-B light (0.5–15 min) at 2°C have the same fluence requirement for chalcone synthase and flavonoid induction as controls irradiated at 25°C. This is indicative of a purely photochemical reaction. Cells fed with riboflavin and irradiated with 6 h of UV-containing white light synthesize higher levels of chalcone synthase and flavonoid than unfed controls. This effect did not occur with blue light. These results indicate that flavin-sensitization requires excitation of flavin and the UV-B light photoreceptor. The in vivo kinetics of flavin uptake and bleaching indicate that the added flavin may act at the surface of the plasma membrane. In view of the likely role of membrane-associated flavin in photoreception, we measured in vitro flavin binding to microsomal membranes. At least one microsomal flavin binding site was solubilized by resuspension of a microsomal pellet in buffer with high KPi and NaCl concentrations and centrifugation at 38000 g. The 38000 g insoluble fraction had much greater flavin binding and contained a receptor with an apparent K_D of about 3.6 μM and an estimated in vivo concentration of at least 6.7 × 10–⁸M. Flavin mononucleotide, roseoflavin, and flavin adenine dinucleotide can compete with riboflavin for this binding site(s), although each has lower affinity than riboflavin. Most microsomal protein was solubilized by resuspension of the microsomal pellet in non-denaturing detergents and centrifugation at 38 000 g ; however, this inhibited flavin binding, presumably because of disruption of the environment of the flavin receptor. The parsley microsomal flavin binding receptor(s) have a possible role in physiological photoreception.     

New technique to trace [<Superscript>15</Superscript>O]water uptake in a living plant with an imaging plate and a BGO detector system

Summary We present a new trial to measure real time water movement in a living plant using the positron emitting radionuclide, ¹⁵O. ¹⁵O was prepared by ¹⁴N(d,n)¹⁵O reaction and 10 ml of ¹⁵O labeled water (2 GBq) was supplied from the root of a soybean plant. To detect activity, an imaging plate (IP) as well as a BGO detector system were used. Since the half-life of ¹⁵O is extremely short, (T_1/2= 122 s), water uptake measurement was performed only for 20 minutes. In order to get [¹⁵O]waterimage, an IP was exposed to the plant for 1 minute for two times. Since the exposure to an IP requires dark condition, a BGO detector system was developed to measure [¹⁵O]waterunder light condition. A couple of BGO probes was set at the lowest stem and the gamma-rays (0.511 MeV) emitted from the radionuclide were measured through coincidence counting and compared with the radioactivity measured from an IP image. Using this system, we have found that the water uptake activity of the plant was drastically reduced under high humidity (99%) and dark condition.     

UVB Radiation Affects Growth,Reproduction and Tissue Structure of Daphnia magna Across Several Temperatures

We examined the effects of daily exposure to UVB on growth, reproduction and histological characteristics of Daphnia magna over two generations at 20, 22, 25 and 30°C. Animals were exposed to 16 h of UVA and photosynthetically active radiation daily. Treated animals received 6 h of UVB during the light phase. Parental (P) generation growth and reproduction was impaired by exposure to UVB at all temperatures, with the poorest production at 30°C. First brood size decreased with UVB exposure; it was lowest at 30°C. Although F1 length at birth increased with P generation age, F1 produced by UVB‐exposed mothers were smaller at all temperatures. The F1 generation was followed at 20 and 25°C; at both temperatures UVB exposure reduced F1 growth and reproduction. F1 growth and F2 production were lowest when both P and F1 generations were exposed to UVB. UVB exposure damaged ovarian and gut tissue at both 25 and 30°C; the consequences of this exposure were more severe at 30°C. The observed tissue damage may relate directly to the UVB‐induced impairment of growth and reproduction. This study provides new insights into the effects of UVB on an important component of the pelagic zooplankton.     

Daphnia pulex Fed UVB-lrradiated Chlamydomonas reinhardtii Show Decreased Survival and Fecundity†

Abstract The ability to avoid or tolerate UVB radiation (290–320 nm) probably reduces the quality of phytoplankton as food for zooplankton. Ultraviolet avoidance forces motile algae lower in the water column, reducing net primary productivity. Production of UV-absorbing compounds as a tolerance mechanism takes carbon and energy away from cell growth; these compounds are also less useful as food for herbivores. We assessed the food quality of UVB-irradiated Chlamydomonas reinhardtii Dang, by analysis of cohort life tables for the herbivore Daphnia pulex Leydig. Algal cultures were grown under fluorescent lights interspersed with UV bulbs (20°C, 16 h light: 8 h dark) and covered with either cellulose acetate (+UVB) or Mylar D (-UVB). After a 7 day UVB exposure, carotenoid levels of C. reinhardtii were significantly greater than in unexposed cultures; there were no significant differences in chlorophyll, lipid, protein or nonstructural carbohydrate levels. After an initial decrease in growth rate, UVB-exposed algae recovered and final growth rates of both cultures were similar after 7 days. Daphnia neonates were raised under optimal conditions (20°C, 16 h light:8 h dark) and fed either UVB-irradiated or control algae. Daphnia fed UVB-irradiated algae had significantly increased mortality, decreased growth rate at the time of first clutch production, delayed release of the first clutch and decreased overall fecundity (P < 0.05). Daphnia were also less efficient at clearing UVB-irradiated algae from the water column (17% removal in 18 h) when compared with Daphnia that grazed upon nonirradiated algae (85% removal in 18 h). These data suggest that even though UVB-irradiated algal populations can recover from their initial decline, their predators may not be able to utilize them as an effective food source. Therefore, the impacts of UVB radiation within     

Effects of Photobiomodulation on Daphnia magna Straus and their Sensitivity to Toxicant

This paper deals with the effect of photobiomodulation (PBM) on Daphnia magna S. and their sensitivity to cadmium sulfate, a known high toxic pollutant. In a first series of experiments, the effect of different He-Ne laser fluences irradiation (range 0.9-4300 mJ cm⁻²) on the fertility of both parent and filial generations (F1–F3) of the crustacean was studied. It was found that PBM in some cases significantly influenced the fertility of both irradiated crustaceans and their nonirradiated offspring. By selecting two fluences (9 ± 2 mJ cm⁻² reducing fertility and 4.3 ± 0.9 J cm⁻² increasing it), the effect of these on toxicity of cadmium sulfate was evaluated. These experiments have shown that prior irradiation with low-intensity light of a helium–neon laser with 632.8 nm wavelength can change the sensitivity of aquatic organisms to toxin cadmium sulfate. The degree and direction of changes depend on the toxicant concentration and the irradiation dose.     

Development of classification models for predicting chronic toxicity of chemicals to Daphnia magna and Pseudokirchneriella subcapitata

Both the acute toxicity and chronic toxicity data on aquatic organisms are indispensable parameters in the ecological risk assessment priority chemical screening process (e.g. persistent, bioaccumulative and toxic chemicals). However, most of the present modelling actions are focused on developing predictive models for the acute toxicity of chemicals to aquatic organisms. As regards chronic aquatic toxicity, considerable work is needed. The major objective of the present study was to construct in silico models for predicting chronic toxicity data for Daphnia magna and Pseudokirchneriella subcapitata. In the modelling, a set of chronic toxicity data was collected for D. magna (21 days no observed effect concentration (NOEC)) and P. subcapitata (72 h NOEC), respectively. Then, binary classification models were developed for D. magna and P. subcapitata by employing the k-nearest neighbour method (k-NN). The model assessment results indicated that the obtained optimum models had high accuracy, sensitivity and specificity. The model application domain was characterized by the Euclidean distance-based method. In the future, the data gap for other chemicals within the application domain on their chronic toxicity for D. magna and P. subcapitata could be filled using the models developed here.     

Comparison of laser ablation-inductively coupled plasma-mass spectrometry and micro-X-ray fluorescence spectrometry for elemental imaging in Daphnia magna

Visualization of elemental distributions in thin sections of biological tissue is gaining importance in many disciplines of biological and medical research. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and scanning micro-X-ray fluorescence spectrometry (micro-XRF) are two widely used microanalytical techniques for elemental mapping. This article compares the capabilities of the two techniques for imaging the distribution of selected elements in the model organism Daphnia magna in terms of detection power and spatial resolution. Sections with a thickness of 10 and 20 μm of the fresh water crustacean Daphnia magna were subjected to LA-ICP-MS and micro-XRF analysis. The elemental distributions obtained for Ca, P, S and Zn allow element-to-tissue correlation. LA-ICP-MS and micro-XRF offer similar limits of detection for the elements Ca and P and thus, allow a cross-validation of the imaging results. LA-ICP-MS was particularly sensitive for determining Zn (LOD 20 μg g⁻¹, 15 μm spot size) in Daphnia magna, while the detection power of micro-XRF was insufficient in this context. However, LA-ICP-MS was inadequate for the measurement of the S distributions, which could be better visualized with micro-XRF (LOD 160 μg g⁻¹, 5 s live time). Both techniques are thus complementary in providing an exhaustive chemical profiling of tissue samples.     

Determination of metallothionein in Daphnia magna by modified square wave cathodic stripping voltammetry

Metallothioneins (MTs) are widely used as biomarkers in environmental studies. However, eliminating interfering factors for MT determination in vivo by electrochemical detection is difficult. In this study, NO₃⁻ and Cl⁻ were found to negatively affect MT determination in Daphnia magna by square wave cathodic stripping voltammetry (SWCSV). The relevant mechanism was analyzed, and results were used to modify the original electrochemical testing parameters and environmental conditions. This modified SWCSV achieved accurate MT concentrations in D. magna, leading to detection limits as low as picomolar levels.     

Photobiological effects of metal ions. Photosensitizing action of uranyl acetate on Escherichia coli K-12

Abstract— The Uranyl acetate sensitized killing of Escherichia coli K-12 by a light source approximating sunlight in both intensity and wavelength distribution is demonstrated to occur at a concentration of 5.0 × 10O^-4M uranyl acetate (pH 7.0). The photosensitized killing was evident after 150 min and almost complete within 320 min. Auxotrophic mutants were isolated from cultures incubated in both light and dark for 160 min at this concentration of uranyl acetate. Binding of UO₂²⁺ to E. coli is shown to occur with 82% of the UO₂²⁺ ions in a 5 × 10^-4M solution (pH 7.0) being bound to the cell wall. In the dark as well as at other pH values the extent of binding was much less. Most of the binding occurred in a time less than 30 min. The observation of rapid binding but delayed photosensitization is attributed to the necessity for penetration of uranyl ions into the cells' interior to effect photosensitization.     

Methiocarb Degradation by Electro-Fenton: Ecotoxicological Evaluation

This paper studies the degradation of methiocarb, a highly hazardous pesticide found in waters and wastewaters, through an electro-Fenton process, using a boron-doped diamond anode and a carbon felt cathode; and evaluates its potential to reduce toxicity towards the model organism Daphnia magna. The influence of applied current density and type and concentration of added iron source, Fe₂(SO₄)₃·5H₂O or FeCl₃·6H₂O, is assessed in the degradation experiments of methiocarb aqueous solutions. The experimental results show that electro-Fenton can be successfully used to degrade methiocarb and to reduce its high toxicity towards D. magna. Total methiocarb removal is achieved at the applied electric charge of 90 C, and a 450× reduction in the acute toxicity towards D. magna, on average, from approximately 900 toxic units to 2 toxic units, is observed at the end of the experiments. No significant differences are found between the two iron sources studied. At the lowest applied anodic current density, 12.5 A m⁻², an increase in iron concentration led to lower methiocarb removal rates, but the opposite is found at the highest applied current densities. The highest organic carbon removal is obtained at the lowest applied current density and added iron concentration.     

Spatial and Temporal Bioactive Compound Contents and Chlorophyll Fluorescence of Kale (Brassica oleracea L.) Under UV-B Exposure Near Harvest Time in Controlled Environments

UV-B irradiation has been used to enhance the secondary metabolite content in plants, but its spatial effect on plants has not been considered. The objective of this study was to compare spatial photosynthetic traits and bioactive compound accumulation in kale (Brassica oleracea L. var Acephala) according to the distribution and length of UV-B exposure near harvest. Plants were exposed to UV-B of 0–3, 3–6 and 6–9 W m⁻² for 4 h per day at 5 days (Exp. 1) and 4.2 W m⁻² at 5, 4, 3, 2 or 1 days (Exp. 2) before harvest. In spatial distribution, the higher the UV-B intensity, the lower the mean F_v/F_m (maximal photochemical efficiency of PSII) and the higher the concentration of total flavonoid compound (TFC). With UV-B stress, F_v/F_m and fluorescence transient parameters decreased except for DI₀/CS (dissipated energy flux per cross section) and PI_abs (performance index of PSII). When exposed to UV-B radiation for 2 days before harvest, the total phenolic compounds and TFC per plant were highest, not always proportional to the local F_v/F_m but affected by dry weight. Short-term UV-B stress near harvest would be more efficient for the accumulation of bioactive compounds by minimizing the loss of plant weight.     

Spectrophotometric investigation of the mechanism of polymerization of isobutylene with vanadium tetrachloride and visible light

The spectra of n-heptane solution of VCl₄, isobutylene, and their mixture at temperatures ranging from +25 to ?80°C in the range of wavelengths from 200 to 2000 nm were investigated. In the region of wavelengths of visible light (400–700 nm) in which the absorption of isobutylene alone and of VCl₄ (concentration lower than 2.2 × 10^?4 mole/l.) is practically zero, their mixtures exhibit an absorption which depends on the concentration of both components and on temperature. A colored complex of VCl₄ and isobutylene is thus obtained, the concentration of which increases with decreasing temperature and is in equilibrium with that of the starting components. The polymerization of isobutylene under the experimental conditions investigated here probably is initiated with the isobutylene–VCl₄ complex after its excitation with light or heat. At low temperatures (t < ?20°C), when the polymerization of isobutylene with VCl₄ virtually does not take place at all in the dark, only excitation with light is operative in the initiation, while at higher temperatures (t > +10°C) thermal excitation plays the predominant role.     

Real-time imaging of ion uptake from root to above-ground part of the plant using conventional beta-ray emitters

We present real-time imaging of ³²P-phosphate within a living plant as well as that in culture solution toward roots. The above-ground part of the plant was irradiated with light from LED and roots were kept in dark. The plant was Lotus japonicus, cv. Miyakojima MG20 and real-time ³²P-phosphate uptake manners in young, flowering and adult phase were visualized. In the case of young plant, there was a preferential translocation of ³²P in younger leaves. When the sample was treated with phosphate deficient condition, ³²P uptake amount was much higher. In an adult phase, ³²P uptake was high at pods.     

Spore Film Dosimeters Are Feasible for UV Dose Monitoring During Heliotherapy

The objective of the study was to compare Bacillus subtilis spore film dosimeters with a Robertson Berger UV meter (RB meter) and diary records for assessing personal UV-B doses during a 13-day heliotherapy (HT) for atopic dermatitis (AD). In addition, the relationship between the personal UV-B dose and change in serum 25-hydroxyvitamin D (25(OH)D) was studied. Altogether 21 adult patients with AD completed the study arranged in the Canary Islands, either in January or March 2005. The spore film dosimeters were used throughout the day during the HT. Serum 25(OH)D was analyzed using radioimmunoassay. The mean personal UV-B dose measured with the dosimeters was 75 SED in January and 131 SED in March. The respective results gained from the RB meter combined with diary records were 63 SED and 119 SED showing a close correlation with the dosimeter results. Serum 25(OH)D concentration increased by 9.7 nmol L⁻¹ in January and by 26.0 7 nmol L⁻¹ in March. The increase in serum 25(OH)D correlated with the UV-B dose received. The patients complied well to use the dosimeters. We conclude spore films to be a feasible and reliable personal UV dosimeter in vivo in field conditions.     

POTENTIAL ERRORS IN THE USE OF CELLULOSE DIACETATE AND MYLAR FILTERS IN UV-B RADIATION STUDIES

Abstract— The increase in UV-B radiation(290–320 nm) penetrating to the earth's surface as a result of the chemical depletion of the stratospheric ozone layer is an important environmental concern. In most studies using artificial UV-B sources, the determination of enhanced UV-B radiation effects on plants relies on equivalent UV-A radiation(320–400 nm) from the experimental UV-B fluorescent lamp source, filtered with either cellulose diacetate (CA) to create UV-B treatments, or with type S Mylar or polyester (PE) to create controls (no UV-B). The spectral irradiance in the UV-A was measured in the dark below lamps at two daily UV-B irradiance levels (14.1 and 10.7 W m^-2) with CA and PE at two ages. Highly significant differences in UV-A radiation (P 0.01) were measured below the treatment/control pairs at both fluence rates and filter ages. Filter aging was observed, which reduced the UV-A irradiance, especially for PE. The total daily ambient UV-A irradiance was also determined in the glasshouse at three seasons: the fall equinox, summer and winter, from which the total daily UV-A (lamp + ambient) irradiances were calculated. The addition of low to moderate ambient irradiance removed the treatment/control differences in the longwave UV-A(350–400 nm); however, the treatment/contro1 differences remained in the shortwave UV-A(320–350 nm), which was restricted by the glass, and in the total UV-A. The treatment/control differences persisted in the shortwave UV-A for the higher irradiance level, even under high summer ambient light. Also, spectral ratios (UVB:UV-A and shortwave: longwave UV-A) for all treatment groups decreased as the ambient UV-A radiation increased. Therefore, a range of experimental conditions exist where PE-covered lamps do not provide adequate control for UV-A irradiance, relative to the CA treatment, for glasshouse/growth chamber experiments. Potential complications in the interpretation of plant response exist for UV-B experiments conducted under low ambient light conditions (e.g. growth chambers; glasshouse in winter) or high daily UV-B irradiances (e.g. 14 kJ m^-2) for those plant responses that are sensitive to UV-A radiation.     

Use of [13C]/[12C] ratios as an indicator of the role of microorganisms in protection of plants from the phytotoxic action of naphthalene

Using molecular and isotopic mass spectrometry, we investigated the toxic effect of naphthalene as a representative of polycyclic aromatic hydrocarbons (PAHs) on plants growing under sterile conditions and plants inoculated with microorganisms capable and incapable of naphthalene degradation. Tobacco plants of the Samsun variety were grown in a closed gas-nutrient system on a mineral medium with sucrose as a carbon source. Naphthalene used as a toxicant at a concentration of 5.2 × 10^?4% contained ¹³C isotope whose amount was characterized by the value δ¹³C = +281.4 ± 0.6‰ relative to the PDB standard and differed from that of sucrose, the main source of carbon (δ¹³C = ?12.0 ± 0.1‰). Degradation of naphthalene was determined by the inclusion of its carbon in metabolic CO₂ and plant tissues (the root, stem, leaves). The effect of naphthalene on plants was indicated by the rates of O₂ production and CO₂ uptake during the light period as compared with the dark period of exposure. A decrease of the toxic effect of naphthalene on plants was observed only at the inoculation of plants with Pseudomonas aureofaciens BS1393 rhizosphere bacteria bearing plasmid pBS216, which controls the naphthalene biodegradation ability. The occurrence of other heterotrophic microorganisms incapable of naphthalene degradation had no similar protective effect.     

A Highly Sensitive Fluorescent Sensor for Palladium and Direct Imaging of Its Ecotoxicity in Living Model Organisms

Rhodamine is an ideal platform for fluorescence probes owing to its spiro‐lactam framework and excellent photochemical properties. Herein, a novel rhodamine‐based palladium fluorescent chemosensor, Rd‐Eb , showing a fast response time (3 min), high sensitivity for palladium species over other ions, and a low detection limit (1.91×10^?7 m ), was synthesized. It can act as an obvious colorimetric as well as a fluorescent “off/on” sensor for Pd²⁺. In addition, it is also an excellent sensor for in vivo imaging of Pd²⁺ in zebra fish and Daphnia magna, illuminating the impact of palladium on organisms at different growth stages with respect to biological toxicology.     

Influence of UV radiation on four freshwater invertebrates

Laboratory tests confirmed a negative and variable response of the following four species to artificial UV radiation: Cypridopsis vidua, an ostracode; Chironomus riparius, a midge larvae; Hyalella azteca, an amphipod; and Daphnia magna, a daphnid. Severe damage occurred at UV-B irradiance ranging from 50 to 80% of incident summer values. Under constant exposure to UV and photosynthetically active radiation (PAR) the acute lethal response was recorded at 0.3, 0.8, 0.8 and 4.9 W m-2 UV-B for D. magna, H. azteca, C. riparius and C. vidua, respectively. Sublethal UV-B damage to invertebrates included impaired movement, partial paralysis, changes in pigmentation and altered water balance (bloating). A series of UV-B, UV-A and PAR treatments, applied separately and in combination, revealed a positive role for both UV-A and PAR in slowing down UV-B damage. Mean lethal concentration values of the species typically more tolerant to UV and PAR (Cypridopsis, Chironomus) decreased conspicuously when both UV-A and PAR were eliminated. For UV-B-sensitive species (Hyalella, Daphnia) these differences were notably smaller. We suggest that this gradation of sensitivity among the tested species demonstrates potential differences in repairing mechanisms which seem to work more efficiently for ostracodes and chironomids than for amphipods and daphnids. Manipulations with a cellulose acetate filter showed that lower range UV-B (280-290 nm), produced by FS-40 lamps, may cause excessive UV damage to invertebrates.     

Comparison of in silico models for prediction of Daphnia magna acute toxicity

Eight in silico modelling packages were evaluated and compared for the prediction of Daphnia magna acute toxicity from the viewpoint of the European legislation on chemicals, REACH. We tested the following models: Discovery Studio (DS) TOPKAT, ACD/Tox Suite, ADMET Predictor?, ECOSAR (Ecological Structure Activity Relationships), TerraQSAR?, T.E.S.T. (Toxicity Estimation Software Tool) and two models implemented in VEGA on 480 industrial compounds for 48-h median lethal concentrations (LC₅₀) to D. magna, matching them with experimental values. The quality of the estimates was compared using a standard statistical review and an additional classification approach in which the hazard predictions were grouped using well-defined regulatory criteria. The regression parameters, correlation coefficient being the most influential, showed that four models (ADMET Predictor?, DS TOPKAT, TerraQSAR? and VEGA DEMETRA) had similar reliability. These performed better than the others, but the coefficient of determination was still low (r² around 0.6), considering that at least half the predicted compounds were inside the training sets. Additionally, we grouped the results in four defined toxicity classes. TerraQSAR? gave 60% of correct classifications, followed by DS TOPKAT, ADMET Predictor? and VEGA DEMETRA, with 56%, 54% and 48%, respectively. These results highlight the challenges associated with developing reliable and easily applied acceptability criteria for the regulatory use of QSAR models to D. magna acute toxicity.