Use of polymorphisms in the γ-gliadin gene of spelt and wheat as a tool for authenticity control

Partial sequencing of the γ-gliadin gene of 62 spelt and 14 soft wheat cultivars was performed. Fifty-six of the 62 spelt cultivars and 13 of the 14 soft wheat cultivars were shown to exhibit the typical spelt or soft wheat γ-gliadin sequence, respectively. Exceptions were ascribed to crossbreeding of soft wheat and spelt. Using the typical soft wheat γ-gliadin sequence, two alternative DNA-based analytical methods were developed for the detection and quantification of spelt flour "adulteration" with soft wheat. A simple and fast detection of soft wheat in spelt flours could be achieved by restriction fragment length (RFLP) analysis. In combination with lab-on-a-chip capillary gel electrophoresis (LOC-CE) the soft wheat proportion could be estimated. Heteroduplex formation served as additional confirmation for the presence of spelt besides soft wheat. Hence, RFLP-LOC-CE constitutes a perfect analysis tool for the quality control of cereal seeds and pure cultivars. A precise quantification of soft wheat "adulterations" in spelt flour down to 1% could be achieved by the developed real-time PCR method. The calibration parameters of the real-time PCR assay fulfilled the minimum performance requirements of the European Network of GMO (genetically modified organisms) Laboratories (ENGL).     

Differentiation of refined and virgin edible oils by means of the trans- and cis-phytol isomer distribution

The differentiation of nonrefined (e.g., cold-pressed) and refined edible oils is an important task in food control because of the higher commercial value of the former. Here, we explored the suitability of the relative abundance of cis-phytol as a marker for authentication of nonrefined edible oils. Phytol, the tetramethyl-branched, monoenoic alcohol, is found widespread in nature as a part of chlorophyll. In chlorophyll, only trans-phytol is found. In this study, we present a method for the analysis of the phytol isomers, considering that traces of cis-phytol (contributing 0.1% to the phytol content) can be determined next to trans-phytol. For this purpose, phytol was gathered with the unsaponifiable matter from the oil, trimethylsilylated, and analyzed by gas chromatography coupled to mass spectrometry. With this method, 27 samples of edible oils (16 refined and 11 nonrefined edible oils) were analyzed for the abundance of cis-phytol relative to trans-phytol. In the nonrefined oils (e.g., olive oil, rapeseed oil, maize oil, and sunflower oil), cis-phytol contributed 0.1% (n = 3) or less (n = 8) to the phytol content. In contrast, the refined olive oils (n = 4) contained a share of 1.3-3% cis-phytol; the refined rapeseed oil (n = 3) contained a share of 0.7-1.0% cis-phytol; and the refined sunflower oil (n = 4) contained a share of 0.3-0.9% cis-phytol. Only one refined pomegranate kernel did not contain cis-phytol. The phytol concentration was not suited to distinguish nonrefined from refined oils. In contrast, our data suggest that the virtual absence of cis-phytol can be used as a marker for nonrefined (e.g., cold-pressed) edible oils.     

Physical properties of a Luvisol for different long‐term fertilization treatments: I. Mesoscale capacity and intensity parameters

The physical properties of a Luvisol derived from loess near Bonn, Germany, under different long‐term fertilization treatments were examined. For the investigation of the impact of farmyard manure (FYM) on soil strength at the mesoscale (100 to 300 cm³ soil cores), undisturbed samples were taken from two different depths (10 and 40 cm), either with no fertilization at all, with full mineral fertilization, with FYM only, and with both mineral and organic fertilization. We investigated hydraulic and mechanical parameters, namely precompression stress, pore‐size distribution, saturated hydraulic and air conductivity, and calculated pore connectivity. Long‐term organic fertilization resulted in significantly more and coarser pores which in addition were more conductant and mechanically stronger by trend. Mineral fertilization also increased pore volume by trend but not pore functionality. Mechanical strength generally increased with fertilization by trend, however, was reduced again when organic and mineral fertilization were combined. Nonetheless, FYM led to relatively higher soil strength as the FYM‐treated plots with lower bulk density attained similar soil strength as the unfertilized but denser plots and thus supported the soil‐improving impact of organic amendments. The subsoil physical properties were rather unaffected by fertilization, but were dominated by texture.     

A combined hydraulic and toxicological approach to assess re-suspended sediments during simulated flood events—part II: an interdisciplinary experimental methodology

Purpose

Flood events are expected to increase both in intensity and frequency due to climate change in the near future. From an environmental toxicology perspective, there is concern that such flood events could lead to the remobilization of contaminated sediment layers in rivers. The aim of this pilot study was to establish a novel and interdisciplinary framework combining methods of hydrodynamic engineering and ecotoxicological assessment to enable investigation of the potential risks associated with such remobilization events.

Materials and methods

Formulated sediment was prepared according to OECD guideline 218 and spiked with a mixture of four polycyclic aromatic hydrocarbons (phenanthrene, chrysene, pyrene, benzo[a]pyrene) at concentrations of 3.3?C8.3?mg?kg^?1 dry weight. Rainbow trout (Oncorhynchus mykiss) were exposed as test animals to re-suspended sediments in three out of five experiments. The experiments were carried out in an annular flume designed to investigate transport behaviour of fine-grained sediments. Several physicochemical (e.g. pH) and sedimentological (e.g. turbidity) parameters were measured to characterise environmental conditions and erosion behaviour of sediments. Furthermore, exposure concentrations were measured by means of an in vitro assay (7-ethoxyresorufin-o-deethylase (EROD), RTL-W1 cell assay) and chemical analysis.

Results and discussion

Preparation and spiking of large amounts of formulated sediments were feasible but not practical. Successful spiking could be confirmed by the bioanalytical methods with the spiked sediments showing significantly elevated EROD induction compared to control sediments. Conditions within the annular flume remained stable throughout all experiments and were adequate to support rainbow trout. Flood events were successfully simulated, resulting in re-suspension of formulated sediment. Different erosion behaviours of sediments during the simulated flood events were observed and could be associated with changes in microbial composition of sediments due to differences in storage conditions. Therefore, maintaining constant storage conditions of formulated sediments is crucial to enable consistency and comparability among erosion experiments.

Conclusions

This study clearly demonstrated the feasibility of a combined hydro-toxicological approach in support of the investigation of the potential ecotoxicological relevance of sediment re-suspension events. However, based on the results presented here, it is recommended to include additional physicochemical parameters, such as redox potential and conductivity, and to extend the experimental setup to natural sediments and different aquatic organisms. Future studies will use natural sediments containing representative microbial communities and extracellular polymeric substances to enable extrapolation from the annular flume to conditions in natural flowing waters.     

Reconciling 14C and minirhizotron‐based estimates of fine‐root turnover with survival functions

The turnover of fine‐roots is a crucial component for the input of C to the soil. The amount of root litter input is depending on estimates of turnover times from different techniques. Turnover times from fine‐root cameras (minirhizotrons) often yield 75% higher root litter input estimates than turnover times estimated with the bomb‐radiocarbon signature of fine roots. We introduce a generic framework for the analysis of fine‐root ¹⁴C with different survival functions. So far, mostly an exponential function has been used to estimate the turnover time and mean age of fine roots. In the context of the introduced survival function framework we clarify the terms turnover time, mean residence time, mean longevity, and mean age, which are commonly used in studies of root turnover. Using a unique time series of fine‐root ¹⁴C (Fröberg 2012), we test if survival functions other than the exponential function are better in accordance with turnover‐time estimates commonly found with other methods. A survival function that corresponds to a two‐pool model was best in agreement with minirhizotron‐based estimates (mean residence time of 1.9 y). We argue that using fine‐root ¹⁴C and minirhizotron time‐to‐death data together would give the best constraints on fine‐root turnover. At the same time this could allow quantifying systematic biases inherent to both techniques.     

The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation

The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(?) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM?, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM? and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.     

The impact of extraction methodologies on the toxicity of sediments in the zebrafish (Danio rerio) embryo test

Purpose

Traditionally, methods for sediment extractions are characterised using chemical analyses. However, in order to evaluate sediment extracts with regard to biological effects and, thus, bioaccessibility, extraction methods have to be compared to effect data obtained from experiments with in situ exposure scenarios, i.e., sediment contact tests. This study compares four extraction methods and sediment contact test data from a previous project with respect to predictive power in the fish embryo test with zebrafish (Danio rerio).

Materials and methods

A natural and an artificial sediment spiked with a mixture of six organic pollutants (2,4-dinitrophenol, diuron, fluoranthene, nonylphenol, parathion and pentachlorophenol) were extracted using (a) membrane dialysis extraction (MDE), (b) a Soxhlet procedure, (c) hydroxypropyl-??-cyclodextrin (HPCD) or (d) Tenax^®-TA. Whereas the former two are regarded being exhaustive with respect to non-covalently bound contaminants, the latter two are considered to predict bioaccessibility. Resulting extracts were tested in the fish embryo assay with D. rerio for embryotoxic and teratogenic potential.

Results and discussion

Mortalities caused by organic extracts from Soxhlet extraction and MDE were high. However, HPCD extracts turned out to be at least as effective as extracts obtained with these two methods. One possible reason might be short ageing time of the spiked sediments. Only Tenax^®-TA extracts gave results comparable to the sediment contact assay for natural sediment, but revealed low reproducibility. Significant differences between natural and artificial sediment were found for extracts obtained with techniques using native (i.e., non-freeze-dried) sediments, i.e., HPCD and Tenax^®-TA. In contrast, MDE and Soxhlet extracts used freeze-dried sediment and did not differentiate between natural and artificial sediment. Therefore, freeze-drying has likely altered and equalised sediment properties that influence accessibility, such as composition of bacterial communities and organic matter quality.

Conclusions

Four extraction methods were successfully characterised with respect to their stringency and predictiveness for bioaccessibility. MDE was confirmed as an alternative to Soxhlet extraction. High mortalities induced by HPCD extracts underline the need to include ageing into consideration when assessing sediments. Although Tenax^®-TA may basically be used to predict bioaccessibility in the fish embryo test, the high variability observed warrants further investigation of the relation between effect and extractability. Apparently, freeze-drying can severely affect sediment properties, potentially eliminating individual properties of natural sediments.     

Identification of isopeptide bonds in heat-treated wheat gluten peptides

Results in this paper confirm heat-induced isopeptide bond formation in wheat gluten. Heating (24 h, 130 °C) of wheat gluten [moisture content 7.4%] decreased its extractability in sodium dodecyl sulfate containing buffer (pH 6.8), even after reduction of disulfide (SS) bonds. Thus, both SS bonds and non-SS bonds were responsible for the extractability loss. Cross-links of the lysinoalanine and lanthionine type were not present in the heated samples, but heat treatment reduced levels of available amino groups. Heating of purified and alkylated high molecular weight glutenin subunits (HMW-GS) under similar conditions also resulted in extractability loss, demonstrating that cross-linking did not solely depend on the availability of cysteine or cystine. These observations indicated that heat treatment had induced isopeptide bond formation, resulting in larger and unextractable molecules. Heating HMW-GS lysine- and glutamine-containing peptides induced the formation of isopeptide bonds, thereby supporting the above hypothesis. The level of isopeptide bond formation increased with heating time.     

蛋氨酸源比较——计算影子价格有助于正确决策

文章通过计算各种蛋氨酸源的影子价格并进行比较,探讨其在实际配方中的应用。以现有肉鸡日粮推荐为基础配制了288个试验日粮,DL-methionine为标准添加蛋氨酸,计算蛋氨酸类似物的影子价格。结果表明:液体蛋氨酸羟基类似物(MHA-FA)的影子价格受多种因素的影响,如果想要替代产品具有竞争力的话,应在假定生物学效价计算出的价格上减去大约5.6%。