ALBERT

Description: Aims Afforestation causes important alterations in SOM content and composition that affect the soil functions and C balance. The aim of this study was to identify the mechanisms that determine the changes in SOM composition following afforestation of grasslands. Methods The study included 4 chronosequences and 5 paired plots comprising pastures and land afforested with Pinus radiata . The SOM was characterized by 13 C CP-MAS NMR spectroscopy and differential scanning calorimetry. Results During the first 10–20 year after afforestation, the changes in SOM content varied from slight gains to large losses (〉40 %). The analyses revealed that even SOM compounds considered resistant to decomposition were degraded during this time. The SOM gains, observed 20 year after stand establishment, were favoured by the higher recalcitrance of pine litter and possibly by soil acidification. The concentrations of most SOM compounds, particularly the stable compounds, were higher at the end of the rotation. The low degree of protection, along with the favourable climatic conditions, may also explain the rapid decomposition of SOM, including resistant compounds, in these soils. DSC analysis complemented the information about SOM composition provided by other techniques. Conclusions The accumulation of stable SOM compounds at the end of the rotation suggests a longer soil C turnover in these afforested soils, which may alleviate the gradual loss of SOC in intensively managed forest soils.

Description: Background and aims Plant nutrient uptake from coarse soil (2–4 mm diameter) has been demonstrated for only a limited number of nutrients, and the nutritional contribution of coarse soil when present with fine soil (material 〈2 mm diameter) in realistic ratios is unknown. We conducted a seedling pot trial to investigate the functional relevance of this soil fraction to plant nutrition. Methods Fine soil was mixed with either coarse soil, or the equivalent volume of inert glass chips, in ratios identical to those occurring naturally in soil sampled from two depths at each of two sites. Seedlings of Nothofagus solandri var. cliffortioides and Weinmannia racemosa were planted in the soil mixtures and harvested after 9 months. Results The content of nitrogen, phosphorous, potassium, magnesium and other elements in the above ground seedling tissue was significantly increased by the presence of coarse soil. The coarse soil fraction also contributed proportionally much more to plant nutrient uptake than fine soil on a mass per mass basis. Conclusions Coarse soil is excluded from conventional soil analysis, so is possible that soil nutrient capital is systematically underestimated. This has implications for land management and studies of plant dynamics in relation to nutrient supply.

Description: Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by reactive oxygen species (ROS), as generated along a variety of biological pathways or through the action of ionizing radiations. In a previous report [ 1 ], we showed that the exposure of cyan fluorescent protein (ECFP) to amounts of • OH that mimic the conditions of intracellular oxidative bursts (associated with intense ROS production) leads to observable changes in its photophysical properties in the absence of any direct oxidation of the ECFP chromophore. In the present work, we analyzed the associated structural modifications of the protein in depth. Following the quantified production of • OH, we devised a complete analytical workflow based on chromatography and mass spectrometry that allowed us to fully characterize the oxidation events. While methionine, tyrosine, and phenylalanine were the only amino acids that were found to be oxidized, semi-quantitative assessment of their oxidation levels showed that the protein is preferentially oxidized at eight residue positions. To account for the preferred oxidation of a few, poorly accessible methionine residues, we propose a multi-step reaction pathway supported by data from pulsed radiolysis experiments. The described experimental workflow is widely generalizable to other fluorescent proteins, and opens the door to the identification of crucial covalent modifications that affect their photophysics. Figure Barrel structure of ECFP: residues that were found to be oxidized by .OH radicals are highlighted

Description: A synthetic redox probe structurally related to natural pyridoacridones was designed and electrochemically characterised. These heterocycles behave as DNA intercalators due to their extended planar structure that promotes stacking in between nucleic acid base pairs. Electrochemical characterization by cyclic voltammetry revealed a quasi-reversible electrochemical behaviour occurring at a mild negative potential in aqueous solution. The study of the mechanism showed that the iminoquinone redox moiety acts similarly to quinone involving a two-electron reduction coupled with proton transfer. The easily accessible potential region with respect to aqueous electro-inactive window makes the pyridoacridone ring suitable for the indirect electrochemical detection of chemically unlabelled DNA. Its usefulness as electrochemical hybridization indicator was assessed on immobilised DNA and compared to doxorubicin. The voltamperometric response of the intercalator acts as an indicator of the presence of double-stranded DNA at the electrode surface and allows the selective transduction of immobilised oligonucleotide hybridization at both macro- and microscale electrodes.

Description: Background and aims Lateral tree-scale variability in plantations should be taken into account when scaling up from point samples, but appropriate methods for sampling and calculation have not been defined. Our aim was to define and evaluate such methods. Methods We evaluated several existing and new methods, using data for throughfall, root biomass and soil respiration in mature oil palm plantations with equilateral triangular spacing. Results Three ways of accounting for spatial variation within the repeating tree unit (a hexagon) were deduced. For visible patch patterns, patches can be delineated and sampled separately. For radial patterns, measurements can be made in radial transects or a triangular portion of the tree unit. For any type of pattern, including unknown patterns, a triangular sampling grid is appropriate. In the case studies examined, throughfall was 79 % of rainfall, with 95 % confidence limits being 62 and 96 % of rainfall. Root biomass and soil respiration, measured on a 35-point grid, varied by an order of magnitude. In zones with steep gradients in parameters, sampling density has a large influence on calculated mean values. Conclusions The methods defined here provide a basis for representative sampling and calculation procedures in studies requiring scaling up from point sampling, but more efficient methods are needed.

Description: The knowledge of RNA’s role in biological systems and the recent recognition of its potential use as a reliable biotherapeutic tool increase the demand for development and validation of analytical methods for accurate analysis of RNA. Affinity chromatography is a unique technique because of the versatility of applications reliant on the affinity ligand used. Recently, an arginine-based matrix has been effectively applied in the purification of RNA because of the specific recognition mechanism for RNA molecules. This interaction is suggested to be due to the length of arginine side chain and its ability to produce good hydrogen bonding geometries, which promote multi-contact with RNA backbone or RNA bases, based on RNA folding. Thus, this work presents the development and validation of an analytical method with ultraviolet detection for the quantification of RNA using affinity chromatography with arginine amino acid as immobilized ligand. The method was validated according to International and European legislation for bioanalytical methods. The results revealed that the proposed method is suitable for the reliable detection, separation, and quantification of RNA, showing that the method is precise and accurate for concentrations up to 200 ng/μL of RNA. Furthermore, the versatility of the methodology was demonstrated by its applicability in the quantification of RNA from different eukaryotic cells and in crude samples of chemically synthesized RNA. Therefore, the proposed method demonstrates a potential multipurpose applicability in molecular biology RNA-based analysis and RNA therapeutics. Figure Proposed interactions occurring between arginine–agarose matrix and RNA molecules. Given the multiplicity of arginine side-chain interactions and depending upon RNA folding state, arginine will preferably bind to phosphate groups of RNA backbone or RNA bases.

Description: Functionalized magnetic nanoparticles have attracted much attention in sample preparation because of their excellent performance compared with traditional sample-preparation sorbents. In this review, we describe the application of magnetic nanoparticles functionalized with silica, octadecylsilane, carbon-based material, surfactants, and polymers as adsorbents for separation and preconcentration of analytes from a variety of matrices. Magnetic solid-phase extraction (MSPE) techniques, mainly reported in the last five years, are presented and discussed.

Description: Racemic mixtures of the promising anti-malarial bisindole alkoids, flinderole A–C, desmethyl flinderole C, borreverine and isoborreverine, are baseline-separated for the first time by HPLC using vancomycin-based stationary phases and partially separated by capillary electrophoresis (CE) using cyclodextrin selectors. The HPLC results compare the performance of Chirobiotic V and V2 in the polar organic and reversed phase modes and their complementary selectivity is discussed. The performance of the cyclodextrin selectors in CE, while less effective, are discussed in terms of their selectivity in normal and reversed polarity modes.

Description: A multidimensional, on-line coupled liquid chromatographic/gas chromatographic system was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs). A two-dimensional liquid chromatographic system (2D-liquid chromatography (LC)), with three columns having different selectivities, was connected on-line to a two-dimensional gas chromatographic system (2D-gas chromatography (GC)). Samples were cleaned up by combining normal elution and column back-flush of the LC columns to selectively remove matrix constituents and isolate well-defined, PAH enriched fractions. Using this system, the sequential removal of polar, mono/diaromatic, olefinic and alkane compounds from crude extracts was achieved. The LC/GC coupling was performed using a fused silica transfer line into a programmable temperature vaporizer (PTV) GC injector. Using the PTV in the solvent vent mode, excess solvent was removed and the enriched PAH sample extract was injected into the GC. The 2D-GC setup consisted of two capillary columns with different stationary phase selectivities. Heart-cutting of selected PAH compounds in the first GC column (first dimension) and transfer of these to the second GC column (second dimension) increased the baseline resolutions of closely eluting PAHs. The on-line system was validated using the standard reference materials SRM 1649a (urban dust) and SRM 1975 (diesel particulate extract). The PAH concentrations measured were comparable to the certified values and the fully automated LC/GC system performed the clean-up, separation and detection of PAHs in 16 extracts in less than 24 h. The multidimensional, on-line 2D-LC/2D-GC system eliminated manual handling of the sample extracts and minimised the risk of sample loss and contamination, while increasing accuracy and precision. Figure Scheme of the 2D-LC/2D-GC system

Description: Recently, an atomic force microscopy (AFM)-based approach for quantifying the number of biological molecules conjugated to a nanoparticle surface at low number densities was reported. The number of target molecules conjugated to the analyte nanoparticle can be determined with single nanoparticle fidelity using antibody-mediated self-assembly to decorate the analyte nanoparticles with probe nanoparticles (i.e., quantitative immunostaining). This work refines the statistical models used to quantitatively interpret the observations when AFM is used to image the resulting structures. The refinements add terms to the previous statistical models to account for the physical sizes of the analyte nanoparticles, conjugated molecules, antibodies, and probe nanoparticles. Thus, a more physically realistic statistical computation can be implemented for a given sample of known qualitative composition, using the software scripts provided. Example AFM data sets, using horseradish peroxidase conjugated to gold nanoparticles, are presented to illustrate how to implement this method successfully.

Description: Background Plants must acquire at least 14 mineral nutrients from the soil to complete their life cycles. Insufficient availability or extreme high levels of the nutrients significantly affect plant growth and development. Plants have evolved a series of mechanisms to adapt to unsuitable growth conditions where nutrient levels are too low or too high. microRNAs (miRNAs), a class of small RNAs, are known to mediate post-transcriptional regulation by transcript cleavage or translational inhibition. Besides regulating plant growth and development, miRNAs are well documented to regulate plant adaptation to adverse environmental conditions including nutrient stresses. Scope In this review, we focus on recent progress in our understanding of how miRNAs are involved in plant response to stresses resulting from deficiency in nutrients, such as nitrogen, phosphorus, sulfur, copper and iron, as well as toxicities from heavy metal ions. Conclusions Accumulated evidence indicates that miRNAs play critical roles in sensing the abundance of nutrients, controlling nutrient uptake and phloem-mediated long-distance transport, and nutrient homeostasis. miRNAs act as systemic signals to coordinate these physiological activities helping plants respond to and survive nutrient stresses and toxicities. Knowledge about how miRNAs are involved in plant responses to nutrient stresses promise to provide novel strategies to develop crops with improved nutrient use efficiency which could be grown in soils with either excessive or insufficient availability of nutrients.

Description: Background and aims (i) compare the concentrations of total polyphenols (TP) and condensed tannins (CT), and CT profiles in different organs of mature trees and seedlings of eight true mangrove species in Hong Kong; (ii) examine the antioxidant activities of CT and (iii) relate the non-enzymatic antioxidative defence system with the vertical zonation pattern of mangrove species. Methods Mature trees and seedlings of eight species were collected from a Hong Kong mangrove swamp to determine TP and CT concentrations and the antioxidant activities of CT. Results According to TP concentrations, the true mangrove species could be broadly classified into three groups, (i) Lumnitzera racemosa and Aegiceras corniculatum 〉 (ii) Heritiera littoralis , Excoecaria agallocha , Bruguiera gymnorrhiza and Kandelia obovata 〉 (iii) Acanthus ilicifolius and Avicennia marina . The last two are pioneer species in the most foreshore location. They also had significantly lower antioxidant activities, CT concentrations and different CT profiles than the other six species in mid- and low-tides. Conclusions Classification of the eight true mangrove species into three groups based on polyphenols was similar to their vertical zonation from land to sea. The relationships between these antioxidants and zonation should be further verified by transplantation studies.

Description: Background and aims Soil phosphorus (P) indices that have been originally developed and applied to agricultural soils for predicting P uptake by plants were examined in a pot experiment to determine the most suitable index for P availability in bauxite-processing residue sand (BRS). Methods Pot trials with ryegrass were established using BRS that had been amended with various organic (greenwaste compost, biochar and biosolids) and inorganic (zeolite) materials and different levels of di-ammonium phosphate fertiliser. Soil P availability indices tested included anion-exchange membrane (AEM-P), 0.01 M calcium chloride (CaCl 2 -P), Colwell-P, and Mehlich 3-P. Results AEM-P was found to most closely reflect the available P status in BRS across all treatments, and had the strongest associations with plant P uptake compared to Colwell-P, Mehlich 3-P and CaCl 2 -P. AEM-P was more closely correlated with P uptake by ryegrass than other P indices, while Colwell-P was closely related to leaf dry matter. Interestingly, a strong inverse relationship between plant indices and pH in BRS growth media was observed, and an adequate level of plant P uptake was found only in 15 year-old rehabilitated BRS with pH 〈 8.0. Conclusions AEM-P was found to be the most suitable index for evaluating P availability in highly alkaline BRS and pH was an important parameter affecting uptake of P by ryegrass. Importantly, time is required (〉 5 years) before improved uptake of P by plants can be observed in rehabilitated residue sand embankments.

Description: Background & aims Herbivore-driven changes to soil properties can influence the decomposition rate of organic material and therefore soil carbon cycling within grassland ecosystems. We investigated how aboveground foraging mammalian and invertebrate herbivores affect mineral soil decomposition rates and associated soil properties in two subalpine vegetation types (short-grass and tall-grass) with different grazing histories. Methods Using exclosures with differing mesh sizes, we progressively excluded large, medium and small mammals and invertebrates from the two vegetation types in the Swiss National Park (SNP). Mineral soil decomposition rates were assessed using the cotton cloth (standard substrate) method between May and September 2010. Results Decomposition displayed strong spatio-temporal variability, best explained by soil temperature. Exclusion of large mammals increased decomposition rates, but further exclusion reduced decomposition rates again in the lightly grazed (tall-grass) vegetation. No difference among treatments was found in the heavily grazed (short-grass) vegetation. Heavily grazed areas had higher decomposition rates than the lightly grazed areas because of higher soil temperatures. Microbial biomass carbon and soil C:N ratio were also linked to spatio-temporal decomposition patterns, but not to grazing history. Conclusions Despite altering some of the environmental controls of decomposition, cellulose decomposition rates in the SNP’s subalpine grasslands appear to be mostly resistant to short-term herbivore exclusion.

Description: Background Our recent publication (Warren et al., Plant Soil 366:683–693, 2013 ) described how pulses of deuterium oxide (D 2 O) or H 2 O combined with neutron radiography can be used to indicate root water uptake and hydraulic redistribution in maize. This technique depends on the large inherent differences in neutron cross-section between D and H atoms resulting in strong image contrast. Scope and Conclusions However, as illustrated by Carminati and Zarebanadkouki ( 2013 ) there can be a change in total water content without a change in contrast simply by a change in the relative proportions of D 2 O and H 2 O. We agree with their premise and detailed calculations (Zarebanadkouki at al. 2012 , 2013 ), and present further evidence that mixing of D 2 O and H 2 O did not confound evidence of hydraulic redistribution in our study.

Description: Background and aims The growth of green plants depends not only on photosynthesis, but also on the successful remobilization and translocation of seed phosphorus (P) reserves to the vegetative parts of the developing seedling during early growth. Remobilization and photosynthesis are therefore two parallel and co-coinciding processes involved in better seedling establishment and early growth. Methods A study was conducted to evaluate the priority of developing maize seedlings to translocate the remobilized seed P reserves and external P uptake to seedling root and shoot sinks during 4 weeks of early growth. Two fluxes of P in growing seedlings, one from seed remobilized P reserves and one from external P uptake, were distinguished by labelling external nutrient solution P with 32 P. Results The seedling phytomass was equally distributed between seedling roots and shoots for 530 cumulated degree days after sowing. Seedlings partitioned up to 71 % of P from seed reserves and up to 68 % of P acquired from the nutrient solution, to the shoots, depending on the seed P content and P concentration in the nutrient solution. It appears that accumulation of P slows down in seedling roots corresponds to the translocative functions of root P towards shoots for start of photosynthesis. Conclusions Our results suggest that the major part of seed P reserves and external P uptake were used in early development of the seedling and the preferred sink was seedling shoots.

Description: Background and aims Nickel (Ni) has become a major heavy metal contaminant. The form of nitrogen nutrition remarkably affects IRT1 expression in roots. IRT1 has an activity of transporting Ni 2+ into root cells. Therefore, nitrogen-form may affect Ni accumulation and toxicity in plants. The assumption was investigated in this study. Methods The Arabidopsis plants were treated in Ni-contained growth solutions with either nitrate (NO 3 − ) or ammonium (NH 4 + ) as the sole N source. After 7-day treatments, Ni concentration, IRT1 expression, Ni-induced toxic symptoms and oxidative stress in plants were analyzed. Results The NO 3 − -fed plants contained a higher Ni concentration, had a greater IRT1 expression in roots, and developed more severe toxic symptoms in the youngest fully expanded leaves, compared with the NH 4 + -fed plants. The Ni-induced growth inhibition was also more significant in NO 3 − -fed plants. Interestingly, Ni exposure resulted in greater hydrogen peroxide (H 2 O 2 ) and superoxide radical (O 2 . − ) accumulations, more severe lipid peroxidation and more cell death in NO 3 − -fed plants, whereas the opposite was true for NH 4 + -fed plants. Furthermore, the Ni-enhanced peroxidase (POD) and superoxide dismutase (SOD) activities were greater in NO 3 − -fed plants Conclusion NO 3 − nutrition promotes Ni uptake, and enhances Ni-induced growth inhibition and oxidative stress in plants compared with NH 4 + nutrition.

Description: Background and aims Brassica napus has high boron (B) demand, but significant genotype differences exist with respect to B deficiency. The aim of this research was to elucidate the relationship between the different sensitivities of Brassica napus cultivars to low B stress and the characteristics of B uptake and transport to characterise the regulation of B efficiency in Brassica napus . Methods B-efficient and B-inefficient Brassica napus cultivars were used to compare the uptake and transport of B using the stable isotope 10 B tracer and grafting experiments, as well as expression of B transporters by RT-PCR. Results B-efficient cultivars have significant advantages with regard to B limitation. The B-efficient cultivar HZ showed less severe B deficiency symptoms and higher dry biomass than the B-inefficient cultivars LW and LB. Both the amount of total B and the 10 B concentration and accumulation in the shoots and roots of B-efficient HZ were higher than those of B-inefficient cultivars. In B-inefficient LW, the amount of total B and the 10 B that was transported into shoots was less than in the other three cultivars and the content and accumulation of total B and 10 B in the roots of B-inefficient LB were the lowest among all of the cultivars. When the roots of B-efficient HZ were used as stocks, the grafted plants showed B-efficient characteristics, such as mild B deficiency symptoms, and higher dry biomass and B accumulation, regardless of whether they originated from B-efficient or B-inefficient cultivars. In contrast, the grafted plants with B-inefficient LW used as stocks were B-inefficient. The expressions of BnBOR1;1c , BnBOR1;2a and BnNIP5;1 were up-regulated in roots under low B stress compared with the normal B condition. However, there was no obvious difference in the expressions of the three genes or of four other BnBOR1s between B-efficient and B-inefficient cultivars in low or normal B environments. Conclusions These results indicate that the B efficiency of Brassica napus is controlled primarily by roots, which allow more uptake and accumulation of B in B-efficient cultivars than B-inefficient cultivars in a low B environment. However the molecular mechanism regulating B efficiency in Brassica napus remains to be determined.

Description: Aims Application of carbon (C) and nitrogen (N) isotopes is an essential tool to study C and N flows in plant-soil-microorganisms systems. When targeting single plants in a community the tracers need to be added via e.g., leaf-labeling or stem-feeding approaches. In this study we: (i) investigated if bicarbonate can be used to introduce 14 C (or 13 C) into white clover and ryegrass, and (ii) compared the patterns of 14 C and 15 N allocation in white clover and ryegrass to evaluate the homogeneity of tracer distribution after two alternative labeling approaches. Methods Perennial ryegrass and white clover were pulse labeled with 15 N urea via leaf-labeling and 14 C either via a 14 CO 2 atm or with 14 C bicarbonate through leaf-labeling. Plants were sampled 4 days after labeling and prepared for bulk isotope analysis and for 14 C imaging to identify plant parts with high and low 14 C activity. Subsequently, plant parts with high and low 14 C activity were separated and analyzed for 15 N enrichment. Results Bicarbonate applied by leaf-labeling efficiently introduced 14 C into both white clover and ryegrass, although the 14 C activity in particular for white clover was found predominantly in the labeled leaf. Using 14 C imaging for identification of areas with high (hotspots) and low 14 C activity showed that 14 C was incorporated very heterogeneously both when using bicarbonate and CO 2 as expected when using pulse labeling. Subsequent analysis of 15 N enrichment in plant parts with high and low 14 C activity showed that 15 N also had a heterogeneous distribution (up to two orders of magnitude). Conclusion Bicarbonate can efficiently be used to introduce 14 C or 13 C into plant via the leaf-labeling method. Both 14 C and 15 N showed heterogeneous distribution in the plant, although the distribution of 15 N was more even than that of 14 C.

Description: Background and aim In numerous areas, rice cultivated under flooded conditions is exposed simultaneously to iron excess and arsenic contamination. The impact of these combined stresses on yield-related parameters and As distribution and speciation in various plant parts remains poorly documented. Methods Rice (cv I Kong Pao) was exposed to iron excess (125 mg L −1 Fe 2 SO 4 ), arsenic (50 and 100 μM Na 2 HAsO 4 .7H 2 O) or a combination of those stressing agents in hydroponic culture until harvest. Plant growth, yield-related parameters, non protein thiols concentration and mineral nutrition were studied in roots and shoots. Arsenic speciation was determined by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. Key Results Iron excess increased As retention by the roots in relation to the development of the root iron plaque but decreased As accumulation in the shoot. Arsenic concentration was lower in the grains than in the shoots. Iron stress reduced As accumulation in the husk but not in the dehusked grains. Iron excess decreased the proportion of extractable As(III) and As(V) in the grain while it increased the proportion of extractable As(III) in the shoot. Combined stresses (Fe+As) affected plant nutrition and significantly reduced the plant yield by limiting the number of grains per plant and the grain filling. Conclusions Fe excess had an antagonist impact on shoot As concentration but an additive negative impact on several yield-related parameters. Iron stress influences both As distribution and As speciation in rice.

Description: Background and aims Intermittently frozen ground in winter is expected to disappear over large areas in the temperate zone due to ongoing climate warming. The lack of soil frost influences plant soil interactions and needs to be studied in more detail. Methods Winter soil frost was avoided by belowground heating wires in a field experiment over two subsequent winters in a temperate grassland. Soil respiration, soil nitrogen availability and plant performance (aboveground biomass, root length at two depth levels, greenness, nutrient content) were compared between “no-frost” and reference plots which underwent repeated freeze-thaw cycles in both winters. Results Soil respiration increased in the “no-frost” treatment during the warming phase (+291 %). N-availability in the upper 10 cm of the soil profile was not affected, possibly due to increased plant N accumulation during winter (+163 %), increased plant N concentration (+18 %) and increased biomass production (+31.5 %) in the growing season. Translocation of roots into deeper soil layers without changes in total root length in response to the “no-frost” treatment, however, may be a sign of nutrient leaching. Conclusions The cumulative effect on carbon cycling due to warmer soils therefore depends on the balance between increased winter carbon loss due to higher soil biotic activity and enhanced plant productivity with higher nutrient accumulation in the growing season.

Description: Aims The aims of this work were to investigate the aluminum (Al) and phosphate (P) interactions in the regulation of root system architecture of Arabidopsis thaliana seedlings and the contribution of auxin signaling in primary and lateral root growth in response to Al toxicity. Methods Detailed analyses of root system architecture and cell division were performed in Arabidopsis WT seedlings and in low phosphorus insensitive mutants lpi1 - 3 and lpr1 - 1 lpr2 - 1 in response to Al. Expression studies of P-deficiency regulated phosphate transporter AtPT2 were also conducted. The role of auxin as a mediator of root morphogenetic changes by Al was evaluated by using the auxin-signaling mutants tir1 , tir1 afb2 afb3 , and arf7 arf19 . Results Al inhibited primary root growth by affecting cell cycle progression and causing differentiation of cells in the root meristem. These effects were reduced in low phosphorus insensitive lpi1 - 3 and low phosphate resistant lpr1 - 1 lpr2 - 1 Arabidopsis mutants. Al also activated the expression of the low phosphate-induced P transporter AtPT2 in roots. Lateral root formation by Al decreased in tir1 afb2 afb3 while arf7 arf19 mutants were highly resistant to Al in both primary root inhibition and lateral root induction. Conclusions Our results suggest that lateral root formation in response to Al toxicity and P deficiency may involve common signaling mechanisms, while a pathway involving ARF7 and ARF19 is important for primary root growth inhibition by Al.

Description: Aims Litter decomposition and subsequent nutrient release play a major role in forest carbon and nutrient cycling. To elucidate how soluble or bulk nutrient ratios affect the decomposition process of beech ( Fagus sylvatica L.) litter, we conducted a microcosm experiment over an 8 week period. Specifically, we investigated leaf-litter from four Austrian forested sites, which varied in elemental composition (C:N:P ratio). Our aim was to gain a mechanistic understanding of early decomposition processes and to determine microbial community changes. Methods We measured initial litter chemistry, microbial activity in terms of respiration (CO 2 ), litter mass loss, microbial biomass C and N (C mic and N mic ), non purgeable organic carbon (NPOC), total dissolved nitrogen (TDN), NH 4 + , NO 3 - and microbial community composition (phospholipid fatty acids – PLFAs). Results At the beginning of the experiment microbial biomass increased and pools of inorganic nitrogen (N) decreased, followed by an increase in fungal PLFAs. Sites higher in NPOC:TDN (C:N of non purgeable organic C and total dissolved N), K and Mn showed higher respiration. Conclusions The C:N ratio of the dissolved pool, rather than the quantity of N, was the major driver of decomposition rates. We saw dynamic changes in the microbial community from the beginning through the termination of the experiment.

Description: Background and aims Grazing may influence nutrient cycling in several ways. In productive mountain grasslands of central Argentina cattle grazing maintain a mosaic of different vegetation patches: lawns, grazed intensively and dominated by high quality palatable plants, and open and closed tussock grasslands dominated by less palatable species. We investigated if differences in the resources deposited on soil (litter and faeces) were associated with litter decomposition rates and soil nitrogen (N) availability across these vegetation patches. Methods We compared the three vegetation patches in terms of litter and faeces quality and decomposability, annual litterfall and faeces deposition rate. We determined decomposition rates of litter and faces in situ and decomposability of the same substrates in a common garden using “litter bags”. We determined soil N availability (with resin bags) in the vegetation patches. Also, we performed a common plant substrates decomposition experiment to assess the effect of soil environment on decomposition process. This technique provides important insights about the soil environmental controls of decomposition (i.e. the sum of soil physicochemical and biological properties, and microclimate), excluding the substrate quality. Results The litter quality and faeces deposition rate were higher in grazing lawns, but the total amounts of carbon (C) and nitrogen (N) deposited on soil were higher in tussock grasslands, due to higher litterfall in these patches. The in situ decomposition rates of litter and faeces, and of the two common plant substrates were not clearly related to either grazing pressure, litterfall or litter quality (C, N, P, lignin, cellulose or hemicellulose content). In situ litter decomposition rate and soil ammonium availability were correlated with the decomposition rates of both common plant substrates. This may suggest that difference in local soil environment among patch types is a stronger driver of decomposition rate than quality or quantity of the resource that enter the soil. Conclusions Our results show that, although high grazing pressure improves litter quality and increases faeces input, the reduction in biomass caused by herbivores greatly reduces C and N input for the litter decomposition pathway. We did not find an accelerated decomposition rate in grazing lawns as proposed by general models. Our results point to soil environment as a potential important control that could mask the effect of litter quality on field decomposition rates at local scale.

Description: Background and aims Cd uptake has been shown to increase during conditions of Fe deficiency. This study tested the hypothesis that Fe-deficiency-responsive genes, particularly OsNRAMP1, play a role in the increased Cd uptake that occurs when rice is grown in aerobic soil conditions. Methods Plants were grown in aerobic or flooded soil conditions. Uptake of Cd was compared to levels of expression of candidate metal transporters and to metal ion availability in soil. Results Plants grown with intermittent soil flooding experienced a predominantly aerobic root environment and had the highest plant Cd uptake. Stronger upregulation of OsNRAMP1 was detected in plants grown in unflooded soil than in flooded soil. However, these transcriptional responses were not linked to an increase in Cd uptake. Overexpression of OsNRAMP1 was not found to increase the uptake of Cd in rice in soil or solution culture. In contrast, there were large differences in availability of Cd, Fe and Mn between flooded and aerobic soils, which were linked to changes in Cd uptake. Conclusions Aerobic soil conditions favour Cd uptake through increased Cd availability and decreased competition between Cd and Fe rather than through the increased expression of the Fe transporters themselves.

Description: Background and aims Intensive land use has led to degradation and abandonment of Portuguese oak woodlands, and subsequent shrub encroachment may have altered the spatial heterogeneity of soil C and N pools. The aim of this study was to evaluate the effects of shrub invasion on soil C and N dynamics in an oak woodland in Southern Portugal. Methods Soil was sampled beneath and outside scattered Quercus suber L. canopies, considering non-encroached areas and areas encroached by shrubs ( Cistus ladanifer L. or Cistus salviifolius L.). Results The spatial heterogeneity of soil C and N contents was mainly associated with tree presence. Outside tree canopies, the labile C pools were larger (mainly beneath C . ladanifer ) and C cycling was faster in encroached areas than in non-encroached areas. Net and gross N mineralization and urease and protease activities were also higher in encroached than in non-encroached areas; however, the metabolic quotient and the Cmicrobial/Corganic ratio were not significantly affected. Beneath the tree canopy, significant effects of encroachment included a small increase in soil labile C and the enzymatic activity beneath C . ladanifer . Conclusions The results indicate the potential capacity of shrub encroachment to accumulate soil organic C in the long term. The rate of soil C and N turnover promoted by shrub encroachment may depend on the Cistus species present.

Description: Aims Decreased expression of TaNAM genes by RNAi results in delayed senescence and decreased grain protein, iron, and zinc concentrations. Here, we determined whether NAM expression level alters onset of senescence under stress conditions, whether delayed senescence in the TaNAM -RNAi line resulted in improved tolerance to post-anthesis abiotic stress, and determined the effects of post-anthesis abiotic stress on N and mineral remobilization and partitioning to grain. Methods Greenhouse-grown WT and TaNAM -RNAi wheat were characterized in two studies:three levels of N fertility or water limitation during grain fill. Studies were conducted under both optimal and heat stress temperatures. Senescence onset was determined by monitoring flag leaf chlorophyll. Results Under optimal tempertures, TaNAM -RNAi plants had a yield advantage at lower N. TaNAM -RNAi plants had delayed senescence relative to the WT and lower grain protein and mineral concentrations, N remobilization efficiency, and partitioning of N and most minerals to grain. Conclusions Nutritional quality of TaNAM -RNAi grain was consistently lower than WT. Delayed senescence of TaNAM -RNAi plants provided a yield advantage under optimal temperatures but not under water or heat stress. Discovery of specific NAM protein targets may allow separation of the delayed senescence and nutrient partitioning traits, which could be used for improvement of wheat.

Description: Aims Nitrification inhibitors (NI) formulated on granulated ammonium sulphate nitrate (ASN) are an option to minimize nitrate leaching into ground waters and emissions of the greenhouse gas N 2 O. This paper focuses (a) on the development of an analytic enabling to extract and quantify the NI 3,4-dimethylpyrazolephosphate (DMPP), marketed since 1999. The efficiency of DMPP has been studied in laboratory and field soils. Here the DMPP analytic and the behaviour of a nitrifying bacterial consortium enriched from a field soil and exposed to zero, field applied and a 10 fold higher DMPP concentration than the recommended one for field application are in the focus. Methods For extracting DMPP quantitatively from soils a method connected to a HPLC analytic has been developed by us and was standardized in laboratory experiment with a silt clay field soil (allochtone Vega). The method is detailed described here. Its reliability has been tested in a 3 years field trial under varying cropping systems and climatic conditions asides the influence of DMPP on CO 2 −, CH 4 − and N 2 O- emissions, measured by the closed chamber method. Parallel a nitrifying bacterial consortium of the silty clay field soil was enriched and subjected to 0, the recommended DMPP concentration for field applications and a 10 times higher one. In incubation experiments the conversion of ammonium to nitrite and nitrate in presence and absence of DMPP was spectrophotometer determined and pH-shifts with a scaled litmus paper. In sacrificed flasks at the end of incubation morphological changes of the bacteria involved were studied by transmission electron microscope (TEM). Results The ammonium, nitrite and nitrate determinations and the TEM pictures show that in presence of the field applied DMPP concentration the nitrifying activity returned around 30 days later than in the control and the cells were slightly enlarged. In presence of a 10 times higher DMPP concentration a recovery was prevented. DMPP prolongs, compared with dicyandiamide (DCD), the period of nitrifiers’ inhibition and reduced N 2 O− and CO 2 − the emissions (Weiske et al., Biol Fertil Soils 34:109–117, 2001a , Nutr Cycl Agroecosys 60:57–64, b ). Conclusions With the method developed by us the stability of DMPP in agricultural soils can be satisfyingly and reproducible studied down to a detection limit of 0.01 μg DMPP g −1 dry soil. The morphological changes in the nitrifying consortium due to DMPP concentrations are in agreement with the recovery rate found by nitrite and nitrate formation.

Description: Background and aims Adequate zinc (Zn) in maize ( Zea mays L.) is required for obtaining Zn-enriched grain and optimum yield. This study investigated the impact of varying Zn fertilizer placements on Zn accumulation in maize plant. Methods Two pot experiments with same design were conducted to investigate the effect of soil Zn heterogeneity by mixing ZnSO 4 ·7H 2 O (10 mg Zn kg −1 soil on an average) in 10–15, 0–15, 25–30, 0–30, 30–60 and 0–60 cm soil layers on maize root growth and shoot Zn content at flowering stage in experiment-1, and assessing effects on grain Zn accumulation at mature stage in experiment-2. Results In experiment-1, Zn placements created a large variation in soil DTPA-Zn concentration (0.3–29.0 mg kg −1 ), which induced a systemic and positive response of root growth within soil layers of 0–30 cm; and shoot Zn content was increased by 102 %–305 % depending on Zn placements. Supply capacity of Zn in soil, defined as sum of product of soil DTPA-Zn concentration and root surface area at different soil layers, was most related to shoot Zn content ( r  = 0.82, P  〈 0.001) via direct and indirect effects according to path analysis. In experiment-2, Zn placements increased grain Zn concentration by up to 51 %, but significantly reduced the grain Zn harvest index from 50 % by control to about 30 % in average. Conclusion Matching the distribution of soil applied Zn with root by Zn placement was helpful to maximize shoot Zn content and grain Zn concentration in maize.

Description: Background and Aims Wetting-drying cycles are important environmental processes known to enhance aggregation. However, very little attention has been given to drying as a process that transports mucilage to inter-particle contacts where it is deposited and serves as binding glue. The objective of this study was to formulate and test conceptual and mathematical models that describe the role of drying in soil aggregation through transportation and deposition of binding agents. Methods We used an ESEM to visualize aggregate formation of pair of glass beads. To test our model, we subjected three different sizes of sand to multiple wetting-drying cycles of PGA solution as a mimic of root exudates to form artificial aggregates. Water stable aggregate was determined using wet sieving apparatus. Results A model to predict aggregate stability in presence of organic matter was developed, where aggregate stability depends on soil texture as well as the strength, density and mass fraction of organic matter, which was confirmed experimentally. The ESEM images emphasize the role of wetting-drying cycles on soil aggregate formation. Conclusions Our experimental results confirmed the mathematical model predictions as well as the ESEM images on the role of drying in soil aggregation as an agent for transport and deposition of binding agents.

Description: Background and aims Many plant growth-promoting endophytes (PGPE) possessing 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity can reduce the level of stress ethylene and assist their host plants cope with various biotic and abiotic stresses. However, information about the endophytic bacteria colonizing in the coastal halophytes is still very scarce. This study aims at isolating efficient ACC deaminase-producing plant growth-promoting (PGP) bacterial strains from the inner tissues of a traditional Chinese folk medicine Limonium sinense (Girard) Kuntze, a halophyte which has high economic and medicinal values grown in the coastal saline soils. Their PGP activity and effects on host seed germination and seedling growth under salinity stress were also evaluated. Methods A total of 126 isolates were obtained from the surface sterilized roots, stems and leaves of L. sinense (Girard) Kuntze. They were initially selected for their ability to produce ACC deaminase as well as other PGP properties such as production of indole-3-acetic acid (IAA), N 2 -fixation, and phosphate-solubilizing activities and subsequently identified by the 16S rRNA gene sequencing. For selected strains, seed germination, seedling growth, and flavonoids production in axenically growth L. sinense (Girard) Kuntze seedlings at different NaCl concentrations (0–500 mM) were quantified. Results Thirteen isolates possessing ACC deaminase activity were obtained. The 16S rRNA gene sequencing analysis showed them to belong to eight genera: Bacillus , Pseudomonas , Klebsiella , Serratia , Arthrobacter , Streptomyces , Isoptericola , and Microbacterium . Inoculation with four of the selected ACC deaminase-producing strains not only stimulated the growth of the host plant but also influenced the flavonoids accumulation. All four strains could colonize and can be re-isolated from the host plant interior tissues. Conclusions These results demonstrate that ACC deaminase-producing habitat-adapted symbiotic bacteria isolated from halophyte could enhance plant growth under saline stress conditions and the PGPE strains could be appropriate as bioinoculants to enhance soil fertility and protect the plants against salt stress.

Description: Aims The objective of this study was to investigate the effects of future warming and drought on (1) the biochemical composition of above-ground biomass of forage plants ( Festuca arundinacea and Dactylis glomerata ), (2) the potential mineralization of this material in soil, and (3) its priming effect on native soil organic matter. Methods We sampled above-ground plant material from spring regrowth and summer regrowth of a climate change experiment. While in spring, the plants were well watered, the summer regrowth was exposed to drought and elevated temperature (+3 °C) by infrared heating of the canopy during 3 weeks. We assessed the elemental and isotopic composition, lignin and non-cellulosic carbohydrate content and composition of plant material grown under all three conditions. Its mineralization potential in soil and priming effects were evaluated during laboratory incubation. Results Warming had no significant effect on elemental and stable isotope composition of both plant materials. In contrast, it resulted in reduction of lignin content for both plant species and decrease of the lignin-to-N ratio for F. arundinacea and increased non-cellulosic carbohydrate content for D. glomerata . Summer regrowth was characterised by increase of δ 13 C values, which is consistent with variations in stomatal conductance due to water shortage. Moreover, summer drought induced an increase in N content leading to decrease of the C/N ratio and increase of lignin-to-N ratio of summer regrowth compared to spring regrowth. Differences in decomposition were small, while priming effects were more strongly altered by the different exposure to enviromental. Conclusion Our results provide direct experimental evidence that extreme climatic events (high temperature and precipitation deficit) have an influence on soil carbon storage particularly through their effect on priming of native soil organic matter induced by altered plant litter. These effects seem to be governed by alterations of stoichiometry and to a smaller extent by alterations of plant chemical composition.

Description: Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2′-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation into regenerating muscle, whereas stem cells labeled in parallel with another dye survived very well and also participated in myofiber formation. Similar data were obtained upon in vitro myogenic culture, and further analysis showed that EdU reduced cell numbers by up to 88 % and increased the cell volume of remaining cells by as much as 91 %. Even at low EdU concentrations, cell survival and phenotype were substantially compromised, and the myogenic differentiation potential was inhibited. Since we examined both primary derived cells and cell lines from several species with the same result, this appears to be a common trait of EdU. We therefore suggest that EdU labeling should be avoided (or used with precaution) for stem cell tracing purposes. Figure Myoblasts were marked with DiI ( red ) and EdU ( purple ), and injected into lesioned skeletal muscle. At day 9 following transplantation, only DiI positive cells were observed and had participated in myofibre formation as (indicated by arrowheads) visualized by red fluorescence signals inside laminin ( green ) positive multinucleated myofibres. EdU was toxic to the engrafted cells, suggesting that this reagent is non-applicaple for tracing of stem cells.

Description: Synthetic cathinones are novel stimulants derived from cathinone, with amphetamines or cocaine-like effects, often labeled “not for human consumption” and considered “legal highs”. Emergence of these new designer drugs complicate interpretation of forensic and clinical cases, with introduction of many new analogs designed to circumvent legislation and vary effects and potencies. We developed a method for the simultaneous quantification of 28 synthetic cathinones, including four metabolites, in urine by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). These cathinones include cathinone, methcathinone, and synthetic cathinones position-3’-substituted, N-alkyl-substituted, ring-substituted, methylenedioxy-substituted, and pyrrolidinyl-substituted. One mL phosphate buffer pH 6 and 25 μL IStd solution were combined with 0.25 mL urine, and subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1 % formic acid in water and in acetonitrile in 20 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by target-MSMS experiments. The assay was linear from 0.5–1 to 100 μg/L, with limits of detection of 0.25–1 μg/L. Imprecision ( n  = 20) was 〈15.9 % and accuracy ( n  = 20) 85.2–118.1 %. Extraction efficiency was 78.9–116.7 % (CV 1.4–16.7 %, n  = 5), process efficiency 57.7–104.9 %, and matrix effects from −29.5 % to 1.5 % (CV 1.9–13.1 %, n  = 10). Most synthetic cathinones were stable at 4 °C for 72 h ( n  = 27) and after 3 freeze-thaw cycles ( n  = 26), but many ( n  = 19) were not stable at room temperature for 24 h (losses up to −67.6 %). The method was applied to authentic urine specimens from synthetic cathinone users. This method provides a comprehensive confirmation method for 28 synthetic cathinones in urine, with good selectivity and specificity.

Description: Amino acid analysis (AAA) has always presented an analytical challenge in terms of sample preparation, separation, and detection. Because of the vast number of amino acids, various separation methods have been applied taking into consideration the large differences in their chemical structures, which span from nonpolar to highly polar side chains. Numerous separation methods have been developed in the past 60 years, and impressive achievements have been made in the fields of separation, derivatization, and detection of amino acids (AAs). Among the separation methods, liquid chromatography (LC) prevailed in the AAA field using either pre-column or post-column labeling techniques in order to improve either separation of AAs or selectivity and sensitivity of AAA. Of the two approaches, the post-column technique is a more rugged and reproducible method and provides excellent AAs separation relatively free from interferences. This review considers current separations combined with post-column labeling techniques for AAA, comparison with the pre-column methods, and the strategies used to develop effective post-column methodology. The focus of the article is on LC methods coupled with post-column labeling techniques and studying the reactions to achieve optimum post-column derivatization (PCD) conditions in order to increase sensitivity and selectivity using various types of detectors (UV–Vis, fluorescence, electrochemical etc.) and illustrating the versatility of the PCD methods for practical analysis. Figure Reaction‐detection scheme for the fluorescent derivative of proline with o‐pthalaldehyde reagent

Description: The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5′-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.

Description: Background and aim Saccharothrix algeriensis NRRL B-24137, isolated from a Saharan soil, has been described as a potential biocontrol agent against Botrytis cinerea and other phytopathogens. However, the plant protection mechanisms involved still need to be described. The aim of this study was to determine this protection phenomenon as well as parts of the mechanisms involved, using Arabidopsis thaliana seedlings and B. cinerea . Methods The bacterial colonization process was evaluated on A. thaliana seedlings using fluorescence in situ hybridization. Protection of A. thaliana seedlings inoculated with NRRL B-24137 against B. cinerea was then evaluated. Parts of the mechanisms involved in the systemic protection against B. cinerea were evaluated using known mutants of genes involved in jasmonate (JA)/ethylene (ET)/salicylic acid (SA) signaling. Other Arabidopsis mutants, AtrhbohD-3 , AtrhbohF-3 , and ups1-1 were also screened to determine other parts of the mechanisms involved. Results The results showed that the strain NRRL B-24137 colonized, epi- and endophytically, the roots of Arabidopsis seedlings but the strain was not a systemic colonizer during the time of the experiment. The strain NRRL B-24137 also reduced B. cinerea symptoms and the protection was linked to known mechanisms of induced systemic resistance (ISR; JA/ET signaling), as well as to functionality of AtrbohF oxidase and of UPS1. Crosstalk between ET/JA and SA signaling could also be involved. Conclusions The isolate NRRL B-24137, after colonizing the root systems of A. thaliana , induces an ISR against B. cinerea , which is JA/ET dependent, but could also require SA crosstalk and protection could also require NAPDH oxidases and UPS1 functionalities.

Description: Background and aims The effect of forest cover distribution and plant litter input on soil organic carbon were analyzed to better understand the dynamics of carbon cycling across ecosystems on the “Natural Oriented Reserve Bosco delle Pianelle”. Fluorescence spectroscopy represents a very useful tool to characterize soil organic matter properties, since it allows to directly monitor the molecular status of a fluorophore depending on its chemical environment, as well as on its structure, substituents of the aromatic moieties, and molecular weight. Here, fluorescence analysis was performed on humic acids isolated from four litters (HALs) and their underlying soils (HAs) at three depths. Methods All samples were collected from a protected forest area, Southern Italy, under different plant covering: Quercus ilex L. (Q), mixed Carpinus betulus L. and Carpinus orientalis Mill. (CC), Pinus halepensis L. (P), and mixed Quercus trojana Webb. and Quercus ilex L. (QQ). Results Data obtained showed a fast decomposition process for P and QQ litters, with HAs in the underlying soils characterized by the presence of simple, highly fluorescent structural components also in the deepest layers. On the contrary, a slow decomposition process was observed for Q and CC litters, whose underlying soil HAs were characterized by an increasing aromatic polycondensation and humification degree from the surface to the deepest layers, as supported by low values of fluorescence intensity and high wavelength maxima. Conclusions Results obtained indicate that P and QQ species promote C accumulation and stock in the underlying soils, thanks to a greater decomposition of their litter, and fluorescence spectroscopy is a very simple and suitable method to evaluate the influence of three species distribution on soil organic carbon pools.

Description: Direct analysis in real time mass spectrometry (DART-MS) has become an established technique for rapid mass spectral analysis of a large variety of samples. DART-MS is capable of analyzing the sample at atmospheric pressure, essentially in the open laboratory environment. DART-MS can be applied to compounds that have been deposited or adsorbed on to surfaces or that are being desorbed therefrom into the atmosphere. This makes DART-MS suitable and well-known for analysis of ingredients of plant materials, pesticide monitoring on vegetables, forensic and safety applications such as screening for traces of explosives, warfare agents, or illicit drugs on luggage, clothes, or bank notes, etc. DART can also be used for analysis of either solid or liquid bulk materials, as may be required in quality control, or to quickly investigate the identity of a compound from chemical synthesis. Even living organisms can be subjected to DART-MS. Driven by different needs in analytical practice, the combination of the DART ionization source and interface can be configured in multiple geometries and with various accessories to adapt the setup as required. Analysis by DART-MS relies on some sort of gas-phase ionization mechanism. In DART, initial generation of the ionizing species is by use of a corona discharge in a pure helium atmosphere which delivers excited helium atoms that, upon their release into the atmosphere, will initiate a cascade of gas-phase reactions. In the end, this results in reagent ions created from atmospheric water or (solvent) vapor in the vicinity of the surface subject to analysis where they effect a chemical ionization process. DART ionization processes may generate positive or negative ions, predominantly even-electron species, but odd-electron species do also occur. The prevailing process of analyte ion formation from a given sample is highly dependent on analyte properties.

Description: In this paper, we report a new type of chiral high-performance liquid chromatography (HPLC) column—a so-called dress-up chiral column—featuring a chiral stationary phase adsorbed reversibly in a commercial fluorous HPLC column through fluorous interactions. We synthesized perfluroalkylated proline derivatives as chiral stationary phase compounds and then adsorbed them reversibly in the fluorous HPLC column through the pumping of their solutions. By using this dress-up chiral column and fluorophobic elution of an aqueous copper(II) sulfate/MeOH mixture, we could enantioseparate seven racemic amino acids within 40 min. When we washed the dress-up chiral column with fluorophilic tetrahydrofuran or MeOH, the adsorbed chiral stationary phase compounds desorbed from the column, completely destroying its enantioseparation ability. The relative standard deviation of the retention times, the number of theoretical plates, and the resolution for each of four preparations of the dress-up columns were all less than or equal to 9.53 % in 20-times repeated analysis, and were all less than or equal to 18.7 % in four different preparations, respectively.

Description: A method for evaluating the interactions between metal ions and nonionic surfactants in aqueous solutions containing high-concentration HCl, using gas pressure-driven low-pressure high-performance liquid chromatography (LP-HPLC) as a highly acid-resistant HPLC system, was developed. To construct the LP-HPLC for this purpose, poly(styrene- co -divinylbenzene)-based low-flow-resistance monolithic columns tolerant to highly acidic conditions were prepared using low-conversion thermal polymerization. Thermal polymerization at 65 °C for 1.5 h (monomer conversions, 33 % for styrene and 59 % for divinylbenzene) allowed preparation of a column with both high separation efficiency (around 60,000 plates m −1 for alkylbenzenes) and a quite low back pressure of 0.14 MPa at a linear flow rate of 1 mm s −1 (2.8 × 10 −13  m 2 in permeability). The base column prepared under the above conditions was coated with a nonionic surfactant, polyoxyethylene nonylphenyl ether (PONPE, average oxyethylene unit numbers ( n ) = 3, 7.5, 15, and 20), and used for evaluation of the interactions between PONPEs and metal ions in 6 M HCl. The interactions between PONPEs and Au(III), Ga(III), Fe(III), Zn(II), and Cu(II) were successfully evaluated using both breakthrough and chromatographic methods. Furthermore, a study of the effect of the polyoxyethylene (POE) chain length revealed that the use of PONPE with the longer POE moiety enhanced the magnitude of the interaction together with the increase in the amount of oxyethylene (OE) units coated on the monolith. Moreover, the interactions of metal ions with a single OE unit were almost constant in the range of n  = 7.5–20, whereas the suppression of the interaction between Au(III) with the shortest PONPE chain ( n  = 3) was also observed. Figure Acid-resistive gas pressure-driven low-pressure high-performance liquid chromatography was developed and applied to the evaluation of interactions between metal ions and nonionic surfactants in high-concentration HCl, in particular for the effect of polyoxyethylene length on the interaction.

Description: In this study, a fast and quantitative determination method for branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, was developed using a pillar array column. A pillar array column with low-dispersion turns was fabricated on a 20 × 20-mm 2 microchip using multistep ultraviolet photolithography and deep reactive ion etching. The BCAAs were fluorescently labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), followed by reversed-phase separation on the pillar array column. The NBD derivatives of the three BCAAs and an internal standard (6-aminocaproic acid) were separated in 100 s. The calibration curves for the NBD-BCAAs had good linearity in the range of 0.4–20 μM, using an internal standard. The intra- and interday precisions were found to be in the ranges of 1.42–3.80 and 2.74–6.97 %, respectively. The accuracies for the NBD-BCAA were from 90.2 to 99.1 %. The method was used for the analysis of sports drink and human plasma samples. The concentrations of BCAAs determined by the developed method showed good agreements with those determined using a conventional high-performance liquid chromatography system. As BCAAs are important biomarkers of some diseases, these results showed that the developed method could be a potential diagnostic tool in clinical research.

Description: The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one, to doping control resulted in hundreds of positive metandienone findings worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methyl steroids, analogs of this metabolite have already been described, but comprehensive characterization and pharmacokinetic data are still missing. In this report, the synthesis of the two epimeric oxandrolone metabolites—17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one—using a fungus ( Cunninghamella elegans ) based protocol is presented. The reference material was fully characterized by liquid chromatography nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete’s urine, different analytical approaches were followed, such as liquid chromatography–tandem mass spectrometry (QqQ and Q-Orbitrap) and gas chromatography–tandem mass spectrometry, in order to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity ( R 2  〉 0.99) was tested, and precise results were obtained for the detection of the analytes (coefficient of variation 〈20 %). Limits of detection (S/N) for confirmatory and screening analysis were estimated at 1 and 2 ng/mL of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods (up to 18 days) for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

Description: Aims The objective of our study was to confirm if hydraulic lift (HL) promotes nutrient uptake in field-grown plants in the same way as demonstrated previously in pot-grown plants. Methods We conducted a field experiment in an agroforestry system, over an entire growing season that included a dry period and a wet period. We used a shallow-root crop plant, mung bean ( Vigna radiata L.), intercropped with walnut ( Juglans regia L.) and jujube ( Zizyphus jujube Mill.), as an indicator for the presence of HL and its effects on nutrient uptake. To monitor HL, we artificially applied deuterium isotope to the deep roots of trees. Results We demonstrated the presence of a natural nitrogen, phosphorus, and potassium gradient along the soil depth, and the occurrence of HL, evidenced by deuterium signature in the shallow soil layers and V. radiata stem, only during the dry season. J. regia and Z. jujube both had deep root systems, but the former species exhibited stronger HL to the shallow soil than the latter. Meanwhile, the upper soil layers of J. regia had significantly higher moisture content, and the intercropped V. radiata had higher nutrient content. Conclusion HL can facilitate water uptake by V. radiata from the upper soil layers in the field condition during the dry season, which relates to nutrient acquisition by the crop.

Description: Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations ( r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D ® ) and a more cost-efficient and transportable planar imaging detector (MAGPIX ® ), hence demonstrating adequate transferability.

Description: Administration of hormonal compounds as growth promoters in livestock farming was banned by Council Directive 96/22/EC. However, this kind of substances is sometimes reported within the framework of European monitoring residue plans. Various analytical methods have been previously developed to screen for their misuse, and they are now especially efficient for monitoring the illegal administration of synthetic and semisynthetic hormones. Nevertheless, proving an exogenous administration of hormones from natural origin (i.e., estradiol-17β or progesterone) still remains a challenge for European authorities. These target compounds are indeed always present in the animal matrix, and the establishment of reference thresholds appears very difficult because of the extreme variability existing among animals. In 2011, a metabolomics study was performed on serum samples obtained from cows treated with estradiol-17β (or its ester estradiol benzoate) and from control animals using a high-performance liquid chromatography (HPLC)-LTQ-Orbitrap system. After appropriate data processing and multivariate statistical analysis (orthogonal partial least squares discriminant analysis), it was possible to highlight one potential biomarker candidate of estradiol treatments in bovine animals. Now, this biomarker has been structurally elucidated as a dipeptide, and its usefulness has been tested through a targeted HPLC-MS/MS method. Its presence in the previous samples has been confirmed and also in additional samples from estradiol-treated animals.

Description: A bootstrapped fuzzy rule-building expert system (FuRES) and a bootstrapped t -statistical weight feature selection method were individually used to select informative features from gas chromatography/mass spectrometry (GC/MS) chemical profiles of basil plants cultivated by organic and conventional farming practices. Feature subsets were selected from two-way GC/MS data objects, total ion chromatograms, and total mass spectra, separately. Four economic classifiers based on the bootstrapped FuRES approach, i.e., fuzzy optimal associative memory (e-FOAM), e-FuRES, partial least-squares–discriminant analysis (e-PLS-DA), and soft independent modeling by class analogy (e-SIMCA), and four economic classifiers based on the bootstrapped t-weight approach, i.e., e-PLS-DA-t, e-FOAM-t, e-FuRES-t, and e-SIMCA-t, were constructed thereafter to be compared with full-size classifiers obtained from the entire GC/MS data objects (i.e., FOAM, FuRES, PLS-DA, and SIMCA). By using three features selected from two-way data objects, the average classification rates with e-FOAM, e-FuRES, e-PLS-DA, and e-SIMCA were 95.3 ± 0.5 %, 100 %, 100 %, and 91.8 ± 0.2 %, respectively. The established economic classifiers were used to classify a new validation set collected 2.5 months later with no parametric change to experimental procedure. Classification rates with e-FOAM, e-FuRES, e-PLS-DA, and e-SIMCA were 96.7 %, 100 %, 100 %, and 96.7 %, respectively. Characteristic components in basil extracts corresponding to highest-ranked useful features were putatively identified. The feature subset may prove valuable as a rapid approach for organic basil authentication.

Description: A small and very simple electromembrane extraction probe (EME-probe) was developed and coupled directly to electrospray ionization mass spectrometry (ESI-MS), and this system was used to monitor in real time in vitro metabolism by rat liver microsomes of drug substances from a small reaction (incubation) chamber (37 °C). The drug-related substances were continuously extracted from the 1.0 mL metabolic reaction mixture and into the EME-probe by an electrical potential of 2.5 V. The extraction probe consisted of a 1-mm long and 350-μm ID thin supported liquid membrane (SLM) of 2-nitrophenyl octyl ether. The drugs and formed metabolites where extracted through the SLM and directly into a 3 μL min −1 flow of 60 mM HCOOH inside the probe serving as the acceptor solution. The acceptor solution was directed into the ESI-MS-system, and the MS continuously monitored the drug-related substances extracted by the EME-probe. The extraction efficiency of the EME-probe was dependant on the applied electrical potential and the length of the SLM, and these parameters as well as the volume of the reaction chamber were set to the values mentioned above to avoid serious depletion from the reaction chamber (soft extraction). Soft extraction was mandatory in order not to affect the reaction kinetics by sample composition changes induced by the EME-probe. The EME-probe/MS-system was used to establish kinetic profiles for the in vitro metabolism of promethazine, amitriptyline and imipramine as model substances.

Description: Aims and background Despite increasing knowledge of the role of allelochemicals in the productivity decline of replanted Chinese fir plantations, relatively little is known about the levels and sources of allelochemicals in relation to autoinhibition. Methods Allelopathic potential of litter, root exudates, and soils in successive rotations of Chinese fir plantations were detected. An allelochemical cyclic dipeptide (6-hydroxy-1,3-dimethyl-8-nonadecyl-[1,4]-diazocane-2,5-dione) from litter, root exudates, and soils in successive rotations was quantified. Results Extracts of leaf litter, fine root, and root exudates significantly inhibited the growth of Chinese fir germinants, and inhibition increased with successive rotations. Similar results were observed in the rhizosphere soil, basal soil, and bulk soil. The largest observed inhibition occurred in the rhizosphere soil. Furthermore, cyclic dipeptide was found in litter, root exudates, and soils, and the concentrations increased with successive rotations. The rhizosphere soil had the highest cyclic dipeptide level, followed by basal soil, while bulk soil contained the lowest concentration. There was a significant positive relationship between the inhibition of radicle growth of Chinese fir germinants and the concentration of cyclic dipeptide. Annual release of cyclic dipeptide through root exudation was 2.08–9.78 mol ha −1 annum, but the annual release of cyclic dipeptide through leaf litter decomposition was lowered to 0.32–1.41 mol ha −1 annum. Conclusions Cyclic dipeptide which caused autoinhibition of Chinese fir may be released into the soil through litter decomposition and root exudation. Root exudates provided more contributions to soil cyclic dipeptide levels than litter in Chinese fir plantations.

Description: Background and aims In the Central Negev hills (Israel) many ancient terraced wadis exist, which captured run-off and caused gradual soil aggradation, which enabled agricultural practices. In these terraces, dark colored soil horizons were observed, containing charcoal, as can be found in Terra Preta soils, suggesting higher fertility compared to natural soils. The aim of our investigation was to investigate these anthropogenic soils and to study the effects of charcoal and ash addition on soil properties and crop growth. Methods We investigated 12 soil profiles, focusing on possible differences between light and dark colored soil horizons. We also investigated the effects of amendment of charcoal and ash on the growth of wheat ( Triticum Aestivum L. ) in a 40-day pot experiment involving two water regimes. Results Results show that charcoal content in light and dark horizons were both low (〈0.2 %), but significantly lower bulk densities were found in dark colored horizons. In the crop experiment, charcoal addition resulted in decreased crop growth, while, in the water deficit regime, ash addition resulted in increased crop growth. Conclusions Considering the observed charcoal and the results from the crop experiment, we hypothesize that, in ancient run-off capturing agricultural systems, ash was purposefully added as fertilizer.

Description: We have recently developed a novel portable NIR imaging device (D-NIRs), which has a high speed and high wavelength resolution. This NIR imaging approach has been developed by utilizing D-NIRs for studying the dissolution of a model tablet containing 20 % ascorbic acid (AsA) as an active pharmaceutical ingredient and 80 % hydroxypropyl methylcellulose, where the tablet is sealed by a special cell. Diffuse reflectance NIR spectra in the 1,000 to 1,600 nm region were measured during the dissolution of the tablet. A unique band at around 1,361 nm of AsA was identified by the second derivative spectra of tablet and used for AsA distribution NIR imaging. Two-dimensional change of AsA concentration of the tablet due to water penetration is clearly shown by using the band-based image at 1,361 nm in NIR spectra obtained with high speed. Moreover, it is significantly enhanced by using the intensity ratio of two bands at 1,361 and 1,354 nm corresponding to AsA and water absorption, respectively, showing the dissolution process. The imaging results suggest that the amount of AsA in the imaged area decreases with increasing water penetration. The proposed NIR imaging approach using the intensity of a specific band or the ratio of two bands combined with the developed portable NIR imaging instrument, is a potentially useful practical way to evaluate the tablet at every moment during dissolution and to monitor the concentration distribution of each drug component in the tablet. Figure Visible photo and NIR image for tablet dissolution obtained by using a newly developed portable NIR imaging device: D-NIRs

Description: Introduction In a recent paper, Warren et al. ( 2013 ) illustrated the potential of neutron radiography to visualize water dynamics in soil and plants. Methods After injection of deuterated water (D 2 O) in soil, the authors could monitor the changes of D 2 O concentration in roots. Results Based on the radiographs, the authors concluded that D 2 O was transported from roots growing in a wet soil region to roots in a dry region, proving hydraulic redistribution between roots. However, this interpretation depends on the correct estimation of D 2 O concentration in soil. Conclusions The experiments of Warren et al. ( 2013 ) could also be explained by diffusion of D 2 O from soil to roots, without hydraulic redistribution within the root system.

Description: Aims The mechanisms of belowground competition are not well understood. Addressing literature reports on competition-induced changes in tree fine root morphology, we conducted a growth experiment with tree saplings to investigate competition effects on important root morphological and functional traits in a root order-focused analysis. Methods European beech and European ash saplings were grown for 34 months in containers under greenhouse conditions in monoculture (2 conspecific plants), in mixture (1 beech and 1 ash) or as single plants. The root system was fractionated according to root orders and eight morphological and functional properties were determined. Results Root order was the most influential factor affecting the fine root traits (except for root diameter and δ 13 C); a significant species identity effect was found for root diameter, tissue density, N concentration and δ 13 C. Ash fine roots were thicker, but had lower tissue densities, contained more N and had systematically higher δ 13 C values than beech roots. The competition treatments had no significant effect on morphological root traits but altered δ 13 C in the 2nd root order. Conclusion Neither intra- nor interspecific root competition affected fine root morphology significantly suggesting that competition-induced root modification may not be a universal phenomenon in temperate trees.

Description: Many in-vitro experiments performed to study the response of thiol-containing proteins to changes in environmental redox potentials use dithiothreitol (DTT) to maintain a preset redox environment throughout the experiments. However, the gradual oxidation of DTT during the course of the experiments, and the interaction between DTT and other components in the system, can significantly alter the initial redox potential and complicate data interpretation. Having an internal reporter of the actual redox potential of the assayed sample facilitates direct correlation of biochemical findings with experimental redox status. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used, well-established tool for analysis and purification of biomolecules, including proteins and peptides. Here, we describe a simple, robust, and quantitative RP-HPLC method we developed and tested for determination of the experimental redox potential of an in-vitro sample at the time of the experiment. It exploits the specific UV-absorbance of the oxidized intrinsic DTT in the samples and retains the high resolving power and high sensitivity of RP-HPLC with UV detection.

Description: The use of smart supports and bioinspired materials to confine living cells and use them for field-deployable biosensors has recently attracted much attention. In particular, bioluminescent whole-cell biosensors designed to respond to different analytes or classes of analyte have been successfully implemented in portable and cost-effective analytical devices. Significant advances in detection technology, biomaterial science, and genetic engineering of cells have recently been reported. Now the challenge is to move from benchtop traditional cell-based assays to portable biosensing devices. Improvement of the analytical performance of these biosensors depends on the availability of optimized bioluminescent reporters, and promising approaches that go beyond reporter gene technology are emerging. To enable handling of cells as ready-to-use reagents, nature-inspired strategies have been used, with the objective of keeping cells in a dormant state until use. Several issues must still be investigated, for example long-term viability of cells, the possibility of performing real-time analysis, and multiplexing capability. Figure Concept of whole-cell bioluminescent biosensor

Description: Background and aims Distinct metal distribution patterns within leaves of metal hyperaccumulating plants are repeatedly observed however, the presumable role of key structural biochemical molecules in determining and regulating their allocation remains largely unknown. We aimed to characterise in a spatially resolved manner the distribution of the main biochemical components in leaves of field-collected Cd/Zn-hyperaccumulating Noccaea praecox in order to relate them to metal distribution patterns at tissue level. Methods The biomolecular composition of the leaves was spatially analysed using synchrotron radiation Fourier Transform Infrared (FTIR) and the distribution of Zn with synchrotron radiation Low-Energy X-Ray Fluorescence (LEXRF) microspectroscopy was determined on the same tissues of interest (epidermis, sub-epidermis, mesophyll). Results In epidermal cells high proportion of free-carboxyl, nitro and phosphate groups standing for pectin, nitroaromatics, phytic and other organic acids were found. Adjacent mesophyll cells had higher proportions of proteins, carbohydrates and cellulosic compounds. Conclusions Pectin compounds were indicated as important components of Zn enriched epidermal cell walls. In addition, intense lignification of epidermal cell walls might limit leakage of the trapped metals back to the metabolically active and thus more sensitive mesophyll. Distribution of metal-binding compounds in particular cell types/tissues may therefore predispose metal distribution patterns and tolerance in leaves.

Description: Aims Tree species affect herb layer species through their effects on soil quality and light regime but their relative importance and interactions are insufficiently known. Methods Pot experiment with soil taken from stands planted with tree species with contrasting effects on soil acidification, two light regimes and six forest perennials. Results The survival or growth of Mercurialis perennis, Lamium galeobdolon , Anemone nemorosa and Primula elatior was lower in the acid Alnus soils than in the less acid Fraxinus soils. By contrast, the acid tolerant Convallaria majalis and Dryopteris dilatata were barely affected by tree species. Light conditions had less impact than soil chemistry and did not compensate for unfavourable soil conditions. Ca and P concentrations increased in plants grown in Fraxinus soils. The Mg and Al shoot/root ratios of respectively one and two of the acid tolerant species was elevated in the most acid soil. Conclusions Tree species effects on forest perennials are mainly explained by increased Al concentrations under acidifying species. Changed plant concentrations and allocation are likely associated to Al antagonism. We found no light compensation for the soil effect on the studied species. However, light alters the plant nutrient concentrations and allocation which may suggest an indirect effect.

Description: A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins were inversely related to the amounts of bound fluorescent labelled mycotoxins (inhibition immunoassay format). The selected monoclonal antibodies were tested for their target mycotoxins and for cross-reactivity with relevant metabolites and masked mycotoxins. In the triplex format, low levels of cross-interactions between the assays occurred at irrelevant high levels only. All three assays were influenced by the sample matrix of cereal extracts to some extent, and matrix-matched calibrations are recommended for quantitative screening purposes. In a preliminary in-house validation, the triplex assay was found to be reproducible, sensitive and sufficiently accurate for the quantitative screening at ML level. The triplex assay was critically compared to liquid chromatography–tandem mass spectrometry using reference materials and fortified blank material. Results for the quantification of ochratoxin A and zearalenone were in good agreement. However, the fumonisin assay was, due to overestimation, only suitable for qualitative judgements. Both flow cytometer platforms (Luminex 100 and FLEXMAP 3D) performed similar with respect to sensitivity with the advantages of a higher sample throughput and response range of the FLEXMAP 3D and lower cost of the Luminex 100. The priciple of the direct inhibition microbead immunoassay using fluorescent mycotoxin-reporter conjugates

Description: Molecularly imprinted polymers (MIPs) are synthetic receptors that are able to specifically bind their target molecules in complex samples, making them a versatile tool in biosensor technology. The combination of MIPs as a recognition element with quartz crystal microbalances (QCM-D with dissipation monitoring) gives a straightforward and sensitive device, which can simultaneously measure frequency and dissipation changes. In this work, bulk-polymerized l -nicotine MIPs were used to test the feasibility of l -nicotine detection in saliva and urine samples. First, l -nicotine-spiked saliva and urine were measured after dilution in demineralized water and 0.1× phosphate-buffered saline solution for proof-of-concept purposes. l -nicotine could indeed be detected specifically in the biologically relevant micromolar concentration range. After successfully testing on spiked samples, saliva was analyzed, which was collected during chewing of either nicotine tablets with different concentrations or of smokeless tobacco. The MIPs in combination with QCM-D were able to distinguish clearly between these samples: This proves the functioning of the concept with saliva, which mediates the oral uptake of nicotine as an alternative to the consumption of cigarettes. Figure Schematics of the sample-preparation procedure for l -nicotine spiked saliva- and urine samples with various concentration levels

Description: Aims The aim of this study was to determine whether goat grazing in the understory of a pine forest at Doñana Natural Park could accelerate the decomposition of the pine needles accumulated on the soil surface and, if so, through which mechanisms. Specifically, the roles of trampling (mechanical fragmentation) and nutrient enrichment through defecation (fertilization) were evaluated in terms of their effect on pine needle decomposition rates. Methods An experiment was conducted featuring the following 4 treatments: 1) intact needles (control), 2) trampled needles, 3) intact needles fertilized with liquid manure, and 4) trampled needles fertilized with liquid manure. Litter decomposition was determined as a function of mass loss over time, using the litter-bag method. Bags were recovered 4, 8, 16, 24 and 36 months after burial in soil, dried and weighed. Needle length, leaf mass per area and C and N concentration were also measured in the buried litter-bags. Results Four months after burial, mass loss was greater in the trampled (23–27 %) than non-trampled (14–16 %) treatments. However, from 8 months onwards, decomposition rates in the fertilized treatments were significantly higher than those in the non-fertilized treatments (between 5 % and 15 % less mass loss). Meanwhile, fertilized treatments presented higher N content (2.1 %) than the non-fertilized ones (1.2 %), with a significantly lower C:N ratio also found in the in the fertilized treatment. Conclusions Trampling and fertilization during grazing accelerates litter decomposition and thus promotes the incorporation of N into the system. Acceleration of decomposition reduces the accumulation of pine needles on the soil surface, reducing the risk of fire.

Description: The generation of key drug metabolites for the purpose of their complete structural characterization, toxicity testing, as well as to serve as standards for quantitative studies, is a critical step in the pharmaceutical discovery and development cycle. Here, we utilized electrochemistry/mass spectrometry for the detection and subsequent generation of six phase I metabolites of simvastatin and lovastatin. Both simvastatin and lovastatin are widely used for the treatment of hypercholesterolemia. There are known drug–drug interaction issues of statin therapy, and it has been suggested that the oxidative metabolites may contribute to the cholesterol-lowering effect of both statins. Of the known phase I metabolites of simvastatin and lovastatin, none are commercially available, and chemical means for the synthesis of a very few of them have been previously reported. Here, we report that electrochemical oxidation of less than 1 mg each of simvastatin and lovastatin led to the generation of three oxidative metabolites of each parent to allow complete nuclear magnetic resonance characterization of all six metabolites. The yields obtained by the electrochemical approach were also compared with incubation of parent drug with commercially available bacterial mutant CYP102A1 enzymes, and it was found that the electrochemical approach gave higher yields than the enzymatic oxidations for the generation of most of the observed oxidative metabolites in this study. Figure Generation of statin drug metabolites by EC/MS (representative mass voltammogram shown), and recombinant CYP enzymes

Description: The binding of a natural anthocyanin to influenza neuraminidase has been studied employing mass spectrometry and molecular docking. Derived from a black elderberry extract, cyanidin-3-sambubiocide has been found to be a potent inhibitor of sialidase activity. This study reveals the molecular basis for its activity for the first time. The anthocyanin is shown by parallel experimental and computational approaches to bind in the so-called 430-cavity in the vicinity of neuraminidase residues 356–364 and 395–432. Since this antiviral compound binds remote from Asp 151 and Glu 119, two residues known to regulate neuraminidase resistance, it provides the potential for the development of a new class of antivirals against the influenza virus without this susceptibility.

Description: A monodisperse molecularly imprinted polymer (MIP) for curcumin was first prepared by precipitation polymerization using methacrylamide (MAM) and 4-vinylpyridine as functional co-monomers, divinylbenzene as a crosslinker, and a mixture of acetonitrile and toluene as a porogen. The use of MAM as the co-monomer resulted in the formation of a monodisperse MIP and non-imprinted polymer (NIP). MIP and NIP, respectively, were monodispersed with a narrow particle size distribution (3.3 ± 0.09 and 3.5 ± 0.10 μm). In addition to shape recognition, hydrophobic and hydrogen-bonding interactions affected the retention and molecular-recognition of curcumin on the MIP. The MIP for curcumin could extract curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in Curcuma longa L . Figure MIPs prepared with 4-VPY ( left hand ) and 4-VPY and MAM ( right hand ) as the functional monomers were polydispersed and monodispersed, respectively

Description: An application of plasma-induced grafting of polyethylene membranes with a thin layer of molecularly imprinted polymer (MIP) was presented. High-density polyethylene (HDPE) membranes, “Vyon,” were used as a substrate for plasma grafting modification. The herbicide atrazine, one of the most popular targets of the molecular imprinting, was chosen as a template. The parameters of the plasma treatment were optimized in order to achieve a good balance between polymerization and ablation processes. Modified HDPE membranes were characterized, and the presence of the grafted polymeric layer was confirmed based on the observed weight gain, pore size measurements, and infrared spectrometry. Since there was no significant change in the porosity of the modified membranes, it was assumed that only a thin layer of the polymer was introduced on the surface. The experiments on the re-binding of the template atrazine to the membranes modified with MIP and blank polymers were performed. HDPE membranes which were grafted with polymer using continuous plasma polymerization demonstrated the best result which was expressed in an imprinted factor equal to 3, suggesting that molecular imprinting was successfully achieved. Figure Atrazine and simazine adsorption by untreated HDPE membranes and membranes plasmagrafted with molecular imprinted polymer

Description: We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase. Figure New zwitterionic ion-exchangers can separate free amino phosphonic acids and a change from Chiralpak ZWIX(+) to ZWIX(-) allows reversal of enantiomer elution order

Description: Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z 31 , with a dissociation constant ( K d ) of 41 ± 5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14 × 10 −9 to 3.14 × 10 −5  M for ZEN, and the detection limit was 7.85 × 10 −10  M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products. Figure Determination of dissociation constant ( K d ) and specificity of aptamers recognizing zearalenone

Description: Countercurrent chromatography (CCC) is an attractive separation method because the analytes are partitioned between two immiscible liquid phases avoiding problems related to solid stationary phase. In recent years, this technique has made great progress in separation power and detection potential. This review describes coupling strategies involving high speed CCC (HSCCC) or centrifugal partition chromatography (CPC). It includes on-line extraction–isolation, hyphenation with mass spectrometry (MS) and nuclear magnetic resonance (NMR) detectors, multidimensional CCC (MDCCC), two-dimensional CCC (2D-CCC), on-line coupling with liquid chromatography (LC), and biological tests, and innovative off-line developments. The basic principles of each method are presented and applications are summarized.

Description: Comprehensive two-dimensional liquid chromatography (LC × LC) has received much attention because it offers much higher peak capacities than separation in a single dimension. The advantageous peak capacity makes it attractive for the separation of complex samples. Various gradient methods have been used in LC × LC systems. The use of continuous shift gradient is advantageous because it combines the peak compression effect of full gradient mode and the tailed gradient program in parallel gradient mode. Here, a comparison of LC × LC analysis of Chinese herbal medicine with full gradient mode and shift gradient mode in the second dimension was performed. A correlation between the first and second dimensions was found in full gradient mode, and this was significantly reduced with shift gradient mode. The orthogonality increased by 43.7 %. The effective peak distribution area increased significantly, which produced better separation.

Description: Background and aims Cadmium (Cd) could activate activity of mitogen-activated protein kinase MPK6 in plants. In this study, we investigated the role of MPK6 in mediating Cd toxicity in plants. Methods The wild type Arabidopsis plants (WT) and the mpk6 - 2 mutants were subjected either 0 (Control) or 10 μM Cd treatment. Kinase activity of MPK6, nitric oxide (NO) level, Cd concentration, and oxidative stress were measured. Results In WT plants, Cd exposure rapidly stimulated kinase activity of MPK6. However, upon Cd exposure, mpk6 - 2 showed better growth than the WT. Although Cd-induced production of NO in roots was greater in WT than in mpk6 - 2 , there was no difference in Cd concentration between the two plants. Nevertheless, the Cd-induced hydroperoxide burst, lipid peroxidation and loss of membrane integrity, were all more severe in the WT than in mpk6 - 2 . Foliar applications of antioxidant ascorbic acid, vigorously improved the growth of both the WT and mpk6 - 2 under Cd exposure. Thereby the growth difference between these two plants was minimized. Conclusions Mutation of mpk6 enhances Cd tolerance in plants by alleviating oxidative stress, but did not affect cadmium accumulation in plants.

Description: Background and Aims The role and linkage of endophytic bacteria to resistance of peanut seeds to biotic stress is poorly understood. The aims of the present study were to survey the experimental (axenic) and control (conventional) peanut plants for the predominant endophytic bacteria, and to characterize isolates with activity against selected A. flavus strains. Methods Young axenic plants were grown from presumably bacteria-free embryos in the lab, and then they were grown in a field. Endophytic bacterial species were identified by the analysis of DNA sequences of their 16S-ribosomal RNA gene. DNA extracted from soil was also analyzed for predominant bacteria. Results Mature seeds from the experimental and control plants contained several species of nonpathogenic endophytic bacteria. Among the eight bacterial species isolated from seeds, and DNA sequences detected in soil, Bacillus thuringiensis was dominant. All B. amyloliquefaciens isolates, the second abundant species in seeds demonstrated activity against A. flavus . This effect was not observed with any other bacterial isolates. There was no significant difference in number and relative occurrence of the two major bacterial species between the experimental and conventionally grown control seeds. Conclusion Endophytic bacterial colonization derives from local soil and not from the seed source, and the peanut plant accommodates only selected species of bacteria from diverse soil populations. Some bacterial isolates showed antibiosis against A. flavus.

Description: Background and Aims Climate warming and increased atmospheric nitrogen (N) deposition both have the potential to increase plant productivity over the next century, yet they can also increase decomposition and respiration. Our aim was to examine the extent to which warming and N addition can, on balance, alter net ecosystem CO 2 exchange (NEE) in a grass-dominated system. Methods We measured NEE responses to warming and N addition over two growing seasons in a temperate old field using steady-state flow-through chambers, which allowed for the integrated measurement of respiration and photoassimilation effects on net CO 2 flux over diel periods. We also assessed the relationship between NEE and plant biomass responses to the warming and N treatments. Results In both years, our study system was a net source of carbon (C) during the snow-free season. N addition did not significantly affect diel NEE or dark respiration in either year, despite a doubling in aboveground plant biomass in response to N addition in the second year, and a corresponding increase in peak daily net CO 2 photoassimilation in N addition plots. The warming treatment also had no significant effect on NEE, although the flow-through chambers required warming to be temporarily halted during NEE measurements. Conclusions Overall, our results both highlight the potential divergence of plant and soil responses to N addition and demonstrate the capacity for a grass-dominated system to function as a net source of C in consecutive years.

Description: Background and aim Intuitively, access to water from the soil at key phenological stages is important for adaptation to drought. This study aimed to assess the temporal pattern of water extraction under terminal drought stress. Methods Pearl millet genotypes with varying levels of terminal drought tolerance were grown in a lysimetric system with a soil volume and plant spacing similar to field conditions. Water extraction was monitored until maturity under differing water regimes. Results The yield did not differ among genotypes under well-watered (WW) conditions, and the water extraction profile of WW plants was similar across all genotypes. In contrast, the yield of sensitive genotypes was 30–100 % lower than that of tolerant lines under water stress (WS). The total volumes of water extracted by tolerant and sensitive genotypes were similar under WS; however, tolerant genotypes extracted less water prior to anthesis, and more water after anthesis. Grain yield was positively related to the amount of water extracted during week three after panicle emergence. Increased water extraction after anthesis benefitted the tillers more than the main culm and was correlated with higher staygreen scores. Conclusion Increased water uptake after anthesis, which results from earlier water conservation during pre-anthesis, increases yield under terminal drought in pearl millet.

Description: Background and aims Efficient accumulation of arsenic (As) in rice ( Oryza sativa L.) poses a potential health risk to rice consumers. The aim of this study was to investigate the mechanisms of uptake, transport and distribution of inorganic arsenic (As i ) and dimethylarsinic acid (DMA) in rice plants. Methods Rice was exposed to As i (As(V)) and DMA in hydroponics. High-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and synchrotron X-ray fluorescence (SXRF) microprobe were used to determine As concentration and the in situ As distribution. Results DMA induced abnormal florets before flowering and caused a sharp decline in the seed setting rate after flowering compared to As i . Rice grains accumulated 2-fold higher DMA than As i . The distribution of As i concentration (root 〉 leaf 〉 husk 〉 caryopsis) in As(V) treatments was different from that of the DMA concentration (caryopsis 〉 husk 〉 root ≥ leaf) in DMA treatments. SXRF showed that As i mainly accumulated in the vascular trace of caryopsis with limited distribution to the endosperm, whereas DMA was observed in both tissues. Conclusions DMA tended to accumulate in caryopsis and induced higher toxicity to the reproductive tissues resulting in markedly reduced grain yield, whereas As i mainly remained in the vegetative tissues and had no significant effect on yield. DMA is more toxic than As i to the reproductive tissues when both of them are at similar concentrations in nutrient solution.

Description: Background and aims Variations in responses to soil N between a non-N-fixing shrub, Baccharis halimifolia L., and a N-fixing shrub, Morella cerifera (L.) Small, were tested over 12 weeks to determine whether N availability is the sole cause of persistent dominance of M. cerifera on barrier islands. Methods Plants were supplied increasing levels of soil N up to 200 mg kg −1 . Measurements included gas exchange and chlorophyll fluorescence parameters across treatments, species, and time. Tissues were analyzed for differences in biomass and nutrients. Results Baccharis halimifolia had reduced physiological responses across all treatment levels, but M. cerifera had comparatively few variations. Across all treatments B. halimifolia photosynthesis and stomatal conductance were reduced by 62 and 76 %, respectively,by week 12. Increasing foliar δ 15 N values across treatments for M. cerifera indicated a shift from utilizing fixed N to available soil N. Biomass was highest at 200 mg kg −1  N for both species. Baccharis halimifolia showed indications of stress response and resource limitation based on physiological responses, nutrient contents, and isotope effects. Conclusions Baccharis halimifolia showed signs of co-limitation of both N and P whereas M. cerifera was limited by neither, suggesting that dominance of M. cerifera is only partially explained by actinorhizal symbiosis and N availability.

Description: Background and Aims Soil mineralization, nitrification, and dynamic changes in abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were studied to validate our hypothesis that soil mineralization and nitrification decreased along the chronosequence of rice cultivation. Methods Paddy soils with a 300, 700 and 2000-year cultivation history (P300, P700 and P2000) were selected to study net mineralization and nitrification processes. Dynamic abundance of AOB and AOA was estimated by quantifying their respective amoA gene copies. Results The net mineralization rate was higher for P300 than P700 and P2000. Potential nitrification ( N p ) and average nitrification rates ( V a ) were similar for P300 and P700 soils, but the simulated potential nitrification rate ( V p ) and nitrification rate (k 1 ) was 72 % and 88 % higher for P300 than P700, respectively. V a was about 70 % lower than for P2000 than P300 and P700. AOB amoA gene copies were higher for P300 than P700 and P2000, whereas AOA abundance did not show significant differences. AOB abundance showed a positive response to NH 4 supply but AOA did not. Conclusions Both N mineralization and nitrification were depressed with increased cultivation time. Archaea responded to mineralization positively rather than nitrification, which suggested that readily mineralized organic matter may play an important role in AOA.

Description: Aims The current study aimed to assess the potential of peanut ( Arachis hypogaea L.) for bioenergy production via phytoextraction in cadmium (Cd) -contaminated soils and screen appropriate cultivars for this approach. Methods A life-cycle pot experiment was conducted to determine the biomass, seed yield, oil content and Cd accumulation of seven peanut cultivars under Cd concentration gradients of 0, 2, and 4 mg kg −1 . Results Peanut exhibits genotypic variations in Cd tolerance, seed production, oil content, and Cd accumulation. Exposure of plants to 2 and 4 mg kg −1 Cd did not inhibit shoot biomass, seed yield, and oil content for most of the cultivars tested. There are large amounts of Cd accumulated in the shoots. Although the seed Cd concentration of peanut was relatively high, the Cd concentration in seed oils was very low (0.04-0.08 mg kg −1 ). Among the cultivars, Qishan 208 showed significant Cd tolerance, high shoot biomass, high pod and seed yield, high seed oil content, considerable shoot Cd concentration, and the largest translocation factor and total Cd in shoots. Conclusions The cultivation of peanut in Cd-contaminated farmland was confirmed to be feasible for bioenergy production via phytoextraction, and Qishan 208 is a good candidate for this approach.

Description: Background and aims Sufficient soil phosphorus (P) is important for achieving optimal crop production, but excessive soil P levels may create a risk of P losses and associated eutrophication of surface waters. The aim of this study was to determine critical soil P levels for achieving optimal crop yields and minimal P losses in common soil types and dominant cropping systems in China. Methods Four long-term experiment sites were selected in China. The critical level of soil Olsen-P for crop yield was determined using the linear-plateau model. The relationships between the soil total P, Olsen-P and CaCl 2 -P were evaluated using two-segment linear model to determine the soil P fertility rate and leaching change-point. Results The critical levels of soil Olsen-P for optimal crop yield ranged from 10.9 mg kg −1 to 21.4 mg kg −1 , above which crop yield response less to the increasing of soil Olsen-P. The P leaching change-points of Olsen-P ranged from 39.9 mg kg −1 to 90.2 mg kg −1 , above which soil CaCl 2 -P greatly increasing with increasing soil Olsen-P. Similar change-point was found between soil total P and Olsen-P. Overall, the change-point ranged from 4.6 mg kg −1 to 71.8 mg kg −1 among all the four sites. These change-points were highly affected by crop specie, soil type, pH and soil organic matter content. Conclusions The three response curves could be used to access the soil Olsen-P status for crop yield, soil P fertility rate and soil P leaching risk for a sustainable soil P management in field.

Description: Background and aims Gaseous losses of ammonia (NH 3 ) have been observed in citrus orchards when urea is surface-applied to the soils, and this loss might significantly limit the effectiveness of the nitrogen (N) fertilizer. However, a portion of the volatilized NH 3 might be absorbed by the plants through the leaves. To quantify the contribution of the leaf absorption of 15 NH 3 , a study with sweet oranges was conducted in two field areas where trees were grown at standard (480 trees ha −1 ) and high densities (617 trees ha −1 ). Methods Plastic trays were filled with soil, covered with mown grass to simulate field management conditions, fertilized with 15 N labeled urea (12 atom % excess) and placed under each of three trees in the orchards. This experimental procedure prevented the uptake of N from the labeled urea by the roots. Two weeks after 15 N fertilization, the trays were removed from the field, and the soil was homogenized and sampled for chemical analyses. The citrus trees under which the trays were placed were destructively harvested, and the total N concentrations and 15 N/ 14 N ratios were determined. Results After urea application, the NH 3 losses peaked within three days and subsequently decreased to negligible amounts after 10 days. The total NH 3 losses accounted for 55–82 % of the applied N. Although the NH 3 absorption by the citrus leaves was proportional to the tree density in the field, only 3–7 % of the 15 NH 3 volatilized from the soil was recovered by the citrus trees, and the NH 3 absorption was also influenced by the proximity of citrus trees to the site of urea application and the leaf areas of the trees. Conclusions The citrus trees can absorb the NH 3 volatilized from urea, even though, the amount recovered by the trees is small and does not represent a significant proportion of total gaseous N losses, what demonstrates the importance of enhanced N use efficiency practices in field to reduce losses of NH 3 when urea is applied to soil surfaces.

Description: Background Rice can accumulate arsenic (As) to relatively high concentrations due to the general flooding practices in rice cultivation, and organic matter in the soil strongly affected As bioavailability to rice plants. The influence of organic matter input on the As transformation in paddy soil and As uptake into rice plants is an area that is rarely investigated. Methods Biogas slurry (BGS), a commonly used organic fertilizer, was applied to an As contaminated paddy soil, in order to investigate the influence of organic matter on As transformation in the paddy soil and As accumulation in rice plants. Results Application of BGS significantly increased the As accumulation in rice plants, especially for methylated As species. Results showed that the concentrations of dissolved organic carbon (DOC) and dissolved Fe(II) in the soil solution were significantly increased by the BGS addition into the paddy soil, and were significantly correlated to the As concentration in the soil solution ( P  〈 0.01). The increase of soil pH and the decrease of the soil redox potential (Eh) were observed as well. These alteration of soil characteristics elevated the As release from soil particles to the soil solution under the addition of BGS. The increased concentrations of dimethylarsinic acid (DMAs(V)) and monomethylarsonic acid (MMAs(V)) in the soil solution, and the volatilized As of trimethylarsine (TMAs) from the paddy soil, suggested that As methylation and volatilization in the soil were also enhanced by BGS addition. The concentrations of methylated As species in rice husks and grains were increased by 105.8–105.9 % and 99.7–112.2 %, respectively. Conclusion These results suggested that the use of organic fertilizer, such as BGS in As-contaminated paddy soil, can significantly alter the behavior of As in soil-rice system and enhance As accumulation in rice plants and should therefore be avoided.

Description: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders resulting from multiple factors. Diagnosis is based on behavioural and developmental signs detected before 3 years of age, and there is no reliable biological marker. The purpose of this study was to evaluate the value of gas chromatography combined with mass spectroscopy (GC-MS) associated with multivariate statistical modeling to capture the global biochemical signature of autistic individuals. GC-MS urinary metabolic profiles of 26 autistic and 24 healthy children were obtained by liq/liq extraction, and were or were not subjected to an oximation step, and then were subjected to a persilylation step. These metabolic profiles were then processed by multivariate analysis, in particular orthogonal partial least-squares discriminant analysis (OPLS-DA, R 2 Y(cum) = 0.97, Q 2 (cum) = 0.88). Discriminating metabolites were identified. The relative concentrations of the succinate and glycolate were higher for autistic than healthy children, whereas those of hippurate, 3-hydroxyphenylacetate, vanillylhydracrylate, 3-hydroxyhippurate, 4-hydroxyphenyl-2-hydroxyacetate, 1 H -indole-3-acetate, phosphate, palmitate, stearate, and 3-methyladipate were lower. Eight other metabolites, which were not identified but characterized by a retention time plus a quantifier and its qualifier ion masses, were found to differ between the two groups. Comparison of statistical models leads to the conclusion that the combination of data obtained from both derivatization techniques leads to the model best discriminating between autistic and healthy groups of children.

Description: Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme , shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination. Figure The Nimzyme high-throughput enzyme activity assay allows for the detection of enzyme activity in cell lysate. Fluorous interactions between a specialized substrate probe and a nanostructure-initiator mass spectrometry surface allow for in situ cleanup and the subsequent collection of unambiguous mass spectra. One of the main hurdles that prevents the widespread adoption of this technology is the need to chemically synthesize the required probes. Here, we present a simplified route to derive Nimzyme probes from a wide variety of biologically interesting substrates.

Description: Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n  = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.

Description: A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C 18 stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae. Figure LC-MS/MS MRM chromatogram of mixed nucleoside and nucleotide standard

Description: Melarsoprol is the only currently available drug for treatment of the late stage of African trypanosomiasis (sleeping sickness). Unfortunately, the arsenic-containing drug causes serious side effects, for which the mechanisms have not been elucidated so far. This investigation describes the study of the melarsoprol biotransformation processes by electrochemical (EC) techniques. Based on EC, potential oxidation reactions of melarsoprol are examined. Moreover, the reactivity of melarsoprol, its metabolite melarsen oxide, and their oxidation products toward the tripeptide glutathione and the proteins hemoglobin and human serum albumin is evaluated. The combination of different analytical techniques allows the identification as well as the quantification of the biotransformation products. The hyphenation of liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI–MS) is applied for identification and structure elucidation, which implies the determination of exact masses and fragmentation patterns. For the selective detection of arsenic containing metabolites, LC coupled to inductively coupled plasma mass spectrometry is utilized. Based on the obtained data, the oxidative biotransformation of melarsoprol can be predicted, revealing novel species which have been suspected, but not been identified up to now. The results of the protein studies prove that melarsen oxide, the active derivative of melarsoprol, strongly binds to human hemoglobin and forms different adducts via the free cysteinyl groups of the hemoglobin α- and β-chain.

Description: Background & aims Plants may have dissimilar effects on ecosystem processes because they possess different attributes. Given increasing biodiversity losses, it is important to understand which plant traits are key drivers of ecosystem functions. To address this question, we studied the response of two ecosystem functions that remove nitrogen (N) from wetland soils, the accumulation of N in plant biomass and denitrification potential (DNP), to variation in plant trait composition. Methods Our experiment manipulated plant composition in a riparian wetland. We determined relative importance of plant traits and environmental variables as predictors of each ecosystem function. Results We demonstrate that Water Use Efficiency (WUE) had a strong negative effect on biomass N. Root porosity and belowground biomass were negatively correlated with DNP. Trait ordination indicated that WUE was largely orthogonal to traits that maximized DNP. Conclusions These results indicate that plant species with different trait values are required to maintain multiple ecosystem functions, and provide a more mechanistic, trait-based link between the recent findings that higher biodiversity is necessary for multi-functionality. While we selected plant traits based on ecological theory, several of the plant traits were not good predictors of each ecosystem function suggesting the ecological theory linking traits to function is incomplete and requires strengthening.

Description: β-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of β-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of β-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by β-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual β-lactam was known. The IC 50 values of 15 β-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 μg L −1 . Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected β-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 μg kg −1 , respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 β-lactams in edible tissues, among which 11 β-lactams controlled by European Union could be detected below maximum residue limits. Figure The receptor-based ELISA for blank sample (negative samples, left ) and sample containing β-lactam antibiotics (positive samples, right )

Description: In recent years, near-infrared (NIR) hyperspectral imaging has proved its suitability for quality and safety control in the cereal sector by allowing spectroscopic images to be collected at single-kernel level, which is of great interest to cereal control laboratories. Contaminants in cereals include, inter alia , impurities such as straw, grains from other crops, and insects, as well as undesirable substances such as ergot (sclerotium of Claviceps purpurea ). For the cereal sector, the presence of ergot creates a high toxicity risk for animals and humans because of its alkaloid content. A study was undertaken, in which a complete procedure for detecting ergot bodies in cereals was developed, based on their NIR spectral characteristics. These were used to build relevant decision rules based on chemometric tools and on the morphological information obtained from the NIR images. The study sought to transfer this procedure from a pilot online NIR hyperspectral imaging system at laboratory level to a NIR hyperspectral imaging system at industrial level and to validate the latter. All the analyses performed showed that the results obtained using both NIR hyperspectral imaging cameras were quite stable and repeatable. In addition, a correlation higher than 0.94 was obtained between the predicted values obtained by NIR hyperspectral imaging and those supplied by the stereo-microscopic method which is the reference method. The validation of the transferred protocol on blind samples showed that the method could identify and quantify ergot contamination, demonstrating the transferability of the method. These results were obtained on samples with an ergot concentration of 0.02 % which is less than the EC limit for cereals (intervention grains) destined for humans fixed at 0.05 %. Online Abstract Figure Pictures showing a the manual removal of ergot bodies and b the observation by the stereo-microscopic method (official method); c the metallic holder with the reference material, and d the NIR hyperspectral SisuCHEMA instrument

Description: Aims Wild soybean accession PI 468917 [ Glycine soja (Sieb. and Zucc.)] was examined for traits that could potentially be beneficial for development of drought resistant soybean cultivars. Methods Water use was examined in controlled environment chambers at three temperatures (25, 30, and 35 °C). Root morphology of plants grown in hydroponics was analyzed using digital imaging software. Results Wild soybean had lower transpiration efficiency in producing mass than the domesticated soybean cultivar Hutcheson at all temperatures. As soil dried, wild soybean decreased transpiration earlier (at a higher soil water content) than domesticated soybean, but only at 25 °C. Wild soybean had much greater root length than the modern soybean when grown at 25 or 30 °C in hydroponics, with the increase observed in the 0.25 to 0.50 mm diameter class. Wild soybean’s advantages dissipated at higher growth temperatures. Conclusions Wild soybean populations, potentially, can offer useful traits for improving drought resistance of modern soybean. Sensitive transpiration control in response to soil drying would contribute to ‘slow-wilting’ strategies known to be advantageous for drought resistance, and greater root length would enhance water acquisition from the soil profile. Use of the traits in breeding programs will require extending the temperature range for trait expression.

Description: Aims Seed germination and seedling emergence are vulnerable to water stress in arid environments. When precipitation is low and unpredictable during the early growing season, seeds near the sand surface often suffer from hydration/dehydration during germination. We investigated the responses of seedling emergence and survival of a sand dune grass with high sand stabilization value to amount and frequency of precipitation and depth of burial in sand. Methods Effects of amount and frequency of precipitation, burial and hydration/dehydration on seedling emergence of Leymus secalinus , were examined using standard procedures. Results Seedling emergence was affected by amount and frequency of monthly precipitation and depth of burial, and it decreased as precipitation frequency decreased with same amount of precipitation. Highest emergence percentage was obtained with 100 or 150 mm precipitation at 1–4 cm depth. Hydration/dehydration treatments decreased germination and increased dormancy percentage. Young seedlings with root lengths of 0–1 mm desiccated up to 30 days revived after rehydration. Conclusions Seedling emergence of L. secalinus is adapted to 150 mm monthly precipitation with frequency of 10–30 times per month, 1–4 cm burial depth and dehydration interval of 1–2 days. Alteration of amount and/or frequency of precipitation caused by climate change could markedly affect seedling emergence and population regeneration of this species.

Description: Background and aims Limited information is available on comparing the iron plaque formation capabilities and their effect on arsenic (As) uptake by different rice plant genotypes grown in As-contaminated soils. This study investigates the effect of iron plaque on As uptake in different rice genotypes grown in As-contaminated soils from the Guandu Plain of northern Taiwan. Methods Twenty-eight rice genotypes including 14 japonica and 14 indica genotypes were used in this study. Rice seedlings were grown in As-contaminated soils for 38 days. The iron plaque formed on the rice roots were extracted using dithionite–citrate–bicarbonate. The concentrations of As, Fe, and P in soil solutions, iron plaque, and plants were measured. The speciation of As in the root’s iron plaque was determined by As K-edge X-ray absorption near-edge structure spectroscopy (XANES). Results The amounts of iron plaque formation on roots were significantly different among 28 tested rice genotypes, and 75.7–92.8 % of As uptake from soils could be sequestered in iron plaque. However, there were no significant negative correlations between the amounts of Fe or As in the iron plaque and the content of As accumulated in rice plants of tested genotypes. XANES data showed that arsenate was the predominant As species in iron plaque, and there were difference in the distribution of As species among different rice genotypes. Conclusions The iron plaque can sequester most of As uptake from soils no matter what rice genotypes used in this study. However, the iron plaque alone did not control the extent of As accumulation in rice plants from As-contaminated soils among 28 tested rice genotypes. Low As uptake genotypes of rice selected from this study can be recommended to be grown in the As-contaminated soils.

Description: Background and aims Freeze events can strongly influence many ecosystem processes. However, the effects of freeze events on litter production, litter quality, and decomposition are rarely documented. Methods In this study, litter fall was measured monthly for 2 years. Two litter decomposition experiments were also performed using freeze-damaged litter and non-damaged litter in a loblolly pine forest. Results The freeze event in November 2009 caused a pronounced pulse of needle litter fall. The freeze-damaged needle litter was shown to have higher N concentration and lower C/N ratio compared with the normal falling needle litter. This finding indicates that freeze damage significantly increased needle quality because of incomplete nutrient resorption. The decomposition of freeze-damaged needle litter was faster than that of normal falling yellow needle litter and slower than that of hand-picked green needle litter. The decomposition rate constant ( k ) was negatively correlated with the C/N ratio in the needle litter. Our results also showed that the different climatic conditions influence patterns of litter decomposition. Conclusions This study suggests that freeze events significantly alter litter quantity and quality, thus affecting litter decomposition rates in a loblolly pine forest in central China.

Description: Background and aims Serpentine soils impose limits on plant growth and survival and thus provide an ideal model for studying plant adaptation under environmental stress. Despite the increasing amount of data on serpentine ecotypic differentiation, no study has assessed the potential role of polyploidy. We tested for links between polyploidy and the response to serpentine stress in Knautia arvensis , a diploid-tetraploid, edaphically differentiated complex. Methods Variation in growth, biomass yield and tissue Mg and Ni accumulation in response to high Mg and Ni concentrations were experimentally tested using hydroponic cultivation of seedlings from eight populations of different ploidy and edaphic origin. Results Regardless of ploidy level, serpentine populations exhibited higher tolerance to both Mg and Ni stress than their non-serpentine counterparts, suggesting an adaptive character of these traits in K. arvensis . The effect of ploidy was rather weak and confined to a slightly better response of serpentine tetraploids to Mg stress and to higher biomass yields in tetraploids from both soil types. Conclusions The similar response of diploid and tetraploid serpentine populations to edaphic stress corresponded with their previously described genetic proximity. This suggests that serpentine tolerance might have been transmitted during the local autopolyploid origin of serpentine tetraploids.