Innovations in the diagnosis of infectious agents of veterinary importance--a seven-league boots fairy tale?

The development of infectiological diagnostic methodology is characterized by saltatoric, revolutionary leaps. Improved sensitivity and specificity, reduction of analytical sample volumes, increased sample throughput, reduced test complexity associated with improved user friendliness are hallmarks of such developments. Apart from purely technical amenities some innovations also enable conceptual adaptations of monitoring strategies. These include the use of diagnostic assays on-site, e. g. in the stable or in the practice. Selecting the assay which is most "fit-for-purpose" among the plethora of sometimes loudly advertised new technology may be difficult even for the well trained and experienced diagnostician. Standardized licensing procedures for infectiological diagnostic kits may be required to guarantee minimal norms of diagnostic quality. At the same time availability of a diversity of diagnostic kits is needed to broaden the base of diagnostic reliability. New infectiological diagnostic innovations will only be useful if the (expanded) results are transposable to the improved practical care of companion animals and farm animal holdings. In addition, the judgment of results, e. g. for epidemiological plausibility, remains a very important issue even when the test results have been produced by novel high technology diagnostics.     

Clusters of hyperactive neurons near amyloid plaques in a mouse model of Alzheimer's disease

The neurodegeneration observed in Alzheimer's disease has been associated with synaptic dismantling and progressive decrease in neuronal activity. We tested this hypothesis in vivo by using two-photon Ca2+ imaging in a mouse model of Alzheimer's disease. Although a decrease in neuronal activity was seen in 29% of layer 2/3 cortical neurons, 21% of neurons displayed an unexpected increase in the frequency of spontaneous Ca2+ transients. These "hyperactive" neurons were found exclusively near the plaques of amyloid beta-depositing mice. The hyperactivity appeared to be due to a relative decrease in synaptic inhibition. Thus, we suggest that a redistribution of synaptic drive between silent and hyperactive neurons, rather than an overall decrease in synaptic activity, provides a mechanism for the disturbed cortical function in Alzheimer's disease.     

Enantioselective analysis of methyl-branched alcohols and acids in rhubarb (Rheum rhabarbarum L.) stalks

The enantiomeric compositions of 2-methylbutanol (1), 4-methylhexanol (2), 2-methylbutanoic acid (3), and 4-methylhexanoic acid (4) present in rhubarb (Rheum rhabarbarum L.) stalks were determined. Enantiodifferentiation was achieved via multidimensional gas chromatography using heptakis(2,3,6-tri-O-ethyl)-beta-cyclodextrin as a chiral stationary phase. For all compounds the enantiomeric ratios were in favor of the (R)-enantiomers. The alcohols (1 and 2) exhibited generally high excesses of the (R)-enantiomers, the ratios varying slightly from batch to batch. For the acid (3) a rather narrow range averaging 65% (R):35% (S) was observed. The procedure applied to isolate the volatiles (vacuum headspace technique, simultaneous distillation--extraction, liquid--liquid extraction) had no significant impact on the enantiomeric ratios. The study describes for the first time a plant used as food material in which 2-methyl-branched volatiles are not nearly exclusively present as (S)-enantiomers. This information enlarges the current regulatory knowledge regarding the classification of these important flavor compounds as "natural" on the basis of their enantiomeric ratios.     

Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation

Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.     

Stereoselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol by lipase-catalyzed hydrolysis of 3-acetylthioesters

The enantioselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol, two potent sulfur-containing aroma compounds, by lipase-catalyzed hydrolysis of the corresponding 3-acetylthioesters was investigated. The stereochemical course of the kinetic resolutions was followed by capillary gas chromatography using modified cyclodextrins as chiral stationary phases. The enzyme preparations tested varied significantly in terms of activity and enantioselectivity (E). The highest E value (E = 36) was observed for the hydrolysis of 3-acetylthiohexanal catalyzed by lipase B from Candida antarctica resulting in (S)-configured thiol products. Immobilization of the enzyme (E = 85) and the use of tert-butyl alcohol as cosolvent (E = 49) improved the enantioselectivity. Modification of the acyl moiety of the substrate (3-benzoylthiohexanal) had no significant impact. The sulfur-containing compounds investigated possess attractive odor properties, and only one of the enantiomers exhibits the pleasant citrus type note.     

GC-based analysis of plant stanyl fatty acid esters in enriched foods

Approaches for the capillary gas chromatographic (GC) based analysis of intact plant stanyl esters in enriched foods were developed. Reference compounds were synthesized by enzyme-catalyzed transesterifications. Their identities were confirmed by means of mass spectrometry. Using a medium polar trifluoropropylmethyl polysiloxane stationary phase, long-chain plant stanyl esters could be separated according to their stanol moieties and their fatty acid chains. Thermal degradation during GC analysis was compensated by determining response factors; calibrations were performed for ten individual plant stanyl esters. For the analysis of low-fat products (skimmed milk drinking yogurts), the GC separation was combined with a "fast extraction" under acidic conditions. For fat-based foods (margarines), online coupled LC-GC offered an elegant and efficient way to avoid time-consuming sample preparation steps. The robust and rapid methods allow conclusions on both, the stanol profiles and the fatty acid moieties, and thus provide a basis for the authentication of this type of functional food ingredients.     

Single immunization with an inactivated vaccine protects sheep from Schmallenberg virus infection

The arthropod-borne Schmallenberg virus (SBV), family Orthobunyaviridae, emerged in Europe in 2011. SBV is associated with a mild disease in adult ruminants but fetal malformation after an infection during a critical phase of pregnancy. A number of inactivated vaccines have been developed; their efficacy after two injections was demonstrated. To make the vaccination of sheep more efficient and economic the effect of a single immunization with one of these vaccines was investigated in the present study. Five vaccinated sheep and five additional control sheep were inoculated with SBV three weeks after vaccination and the results of a competitive ELISA, a standard microneutralization test and an SBV-specific real-time RT-PCR confirmed vaccine efficacy by demonstrating complete inhibition of viral replication in immunized animals.     

Simultaneous analysis of free phytosterols/phytostanols and intact phytosteryl/phytostanyl fatty acid and phenolic acid esters in cereals

An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed in their intact form by means of capillary gas chromatography. Besides free sterols and stanols, up to 33 different fatty acid and phenolic acid esters were identified in four different cereal grains via gas chromatography-mass spectrometry. The majority (52-57%) of the sterols and stanols were present as fatty acid esters. The highest levels of all three sterol and stanol classes based on dry matter of ground kernels were determined in corn, whereas the oil extract of rye was 1.7 and 1.6 times richer in fatty acid esters and free sterols/stanols than the corn oil. The results showed that there are considerable differences in the sterols/stanols and their ester profiles and contents obtained from corn compared to rye, wheat, and spelt. The proposed method is useful for the quantification of a wide range of free phytosterols/phytostanols and intact phytosteryl/phytostanyl esters to characterize different types of grain.     

Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 1. Assessing analytical validation

Current tools used to assess the safety of food and feed derived from modern biotechnology emphasize the investigation of possible unintended effects caused directly by the expression of transgenes or indirectly by pleiotropy. These tools include extensive multisite and multiyear agronomic evaluations, compositional analyses, animal nutrition, and classical toxicology evaluations. Because analytical technologies are rapidly developing, proteome analysis based on two-dimensional gel electrophoresis (2DE) was investigated as a complementary tool to the existing technologies. A 2DE method was established for the qualitative and quantitative analysis of the seed proteome of Arabidopsis thaliana with the following validation parameters examined: (1) source and scope of variation; (2) repeatability; (3) sensitivity; and (4) linearity of the method. The 2DE method resolves proteins with isoelectric points between 4 and 9 and molecular masses (MM) of 6-120 kDa and is sensitive enough to detect protein levels in the low nanogram range. The separation of the proteins was demonstrated to be very reliable with relative position variations of 1.7 and 1.1% for the pI and MM directions, respectively. The mean coefficient of variation of 254 matched spot qualities was found to be 24.8% for the gel-to-gel and 26% for the overall variability. A linear relationship (R2 > 0.9) between protein amount and spot volume was demonstrated over a 100-fold range for the majority of selected proteins. Therefore, this method could be used to interrogate proteome alterations such as a novel protein, fusion protein, or any other change that affects molecular mass, isoelectric point, and/or quantity of a protein.     

Influence of the distillation step on the ratios of stable isotopes of ethanol in cherry brandies

Isotope ratio mass spectrometry and site-specific natural isotope fractionation-nuclear magnetic resonance were applied to determine the overall carbon isotope ratio (delta13C) and the hydrogen isotope ratios [(D/H)I and (D/H)II] of ethanol, respectively. Ethanol was obtained by distillation of fermented cherry mash from a pot still commonly used in fruit brandy production. Analyses of distillate fractions revealed that the distillation proceeds with a fractionation of ethanol isotopologues. The inverse vapor pressure isotope effect (VPIE) observed for the carbon isotopologues is in accordance with the data reported for distillation of ethanol in spinning band columns. In contrast, the inverse VPIE for hydrogen isotopologues of ethanol observed in spinning band columns could not be confirmed. To investigate whether the observed isotope fractionations might influence the applicability of stable isotope analysis for quality and authenticity assessment of fruit brandies, the collected distillate fractions were recombined to cuts, as is common practice in commercial fruit brandy production. Taking into consideration the limits of repeatability of the method, it could be demonstrated that the isotope fractionations observed do not impair the applicability of stable isotope analysis of the carbon and hydrogen isotopes of ethanol for the authenticity assessment of cherry brandies if the cuts are placed in accordance with common distillers' practice.