Micro-scaled high-throughput digestion of plant tissue samples for multi-elemental analysis

Background  

Quantitative multi-elemental analysis by inductively coupled plasma (ICP) spectrometry depends on a complete digestion of solid samples. However, fast and thorough sample digestion is a challenging analytical task which constitutes a bottleneck in modern multi-elemental analysis. Additional obstacles may be that sample quantities are limited and elemental concentrations low. In such cases, digestion in small volumes with minimum dilution and contamination is required in order to obtain high accuracy data.     

Hepatic lipidosis: Liver characteristics and acute phase proteins in affected turkeys

The hepatic lipidosis (HL) in fattening turkeys is a disease has been known for a long time, but the cause and pathogenesis is still not clarified. A recent study reported unexplained high levels of iron in liver tissue of fattening turkeys suffering from HL. In this study, the iron status, possible infectious or inflammatory influences in form of an acute phase reaction and the analysis of fatty acid pattern in liver tissue of turkeys affected by HL were examined. Three cases of HL on three different fattening turkey farms were investigated during the outbreak of the disease. Clinically affected and non-affected animals were subjected to a pathological examination, where the diagnosis HL or non-affected was made. In total, 70 birds were examined (40 with HL, 30 without HL) and blood and liver samples were taken. Additionally, samples from 15 slaughtered birds were taken as a further control group. In liver tissue, the iron content and the content of long-chain fatty acids were determined; in blood samples, ferritin and transferrin were measured. The iron content in liver tissue was more than three times higher for animals with HL than among non-affected animals and the control group. The transferrin levels were lowest for animals with HL, highest in the control group and in between for non-affected animals. The fatty acid pattern in liver tissue of affected animals indicated a shift from polyunsaturated fatty acids to saturated fatty acids and monounsaturated fatty acids compared to the control group and the non-affected animals. Overall, the non-affected animals of a flock affected by HL were similar to the healthy animals of the abattoir. The low acute phase protein levels for animals with HL together with high iron contents could indicate a previous malnutrition/starvation period and/or severe liver damage for those animals suffering from HL.     

Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]⁺ ions.     

The influence of diet on Lawsonia intracellularis colonization in pigs upon experimental challenge

The objective of this investigation was to study if different feeding strategies influence experimental infections of pigs with Lawsonia intracellularis, the causative agent of proliferative enteropathy. In three sequential trials, a total of 144 weaned pigs were fed five different diets all made from a standard diet based on wheat and barley as carbohydrate source and soybean as protein source. The five diets were: a standard diet (fine ground and pelleted), the standard diet fed as fermented liquid feed, the standard diet added 1.8% formic acid, the standard diet added 2.4% lactic acid and a diet similar to the standard diet (made from the same ingredients), but fed coarse ground. Twenty-four pigs on each diet were orally inoculated with L. intracellularis and growth performance and faecal excretion of bacteria were monitored. Twenty-four pigs fed the standard diet were included as not experimentally infected controls. Pigs in the first two trials were sacrificed 4 weeks post-inoculation, whereas animals in the third trial were sacrificed after 5 weeks. Pigs in all experimentally infected groups excreted L. intracellularis. The fermented liquid diet delayed the excretion of L. intracellularis and furthermore, pigs fed the standard diet supplemented with lactic acid had limited pathological lesions when the intestines were examined 4 weeks after inoculation. The growth performance was reduced in pigs experimentally challenged with L. intracellularis, however the prevalence and severity of diarrhea was limited.     

Incomplete split-plot designs based on α-designs: a compromise between traditional split-plot designs and randomised complete block design

The paper shows how the α-design (also known as generalised lattice) may be used for constructing incomplete split-plot designs and describes four different methods (A, B, C and D) of construction. Intra-block efficiency factors and theoretical considerations are used to compare the methods. Based on those considerations method B was considered to be the most appropriate method for trials where tests for interaction between the two factors were important and thus this method was used and most of the paper deals with trials based on this construction method. The incomplete split-plots were superior to traditional split-plots in most cases—and the increase in efficiency of the designs can be quite large—especially for comparisons involving the whole-plot treatment. The efficiency for the comparison of the main effect of the whole-plot treatment was in most cases larger for randomized complete block design than for the incomplete split-plot design, but for other comparisons the proposed designs were in most cases more efficient than a randomized complete block design. The efficiency of the designs was compared to traditional split-plot designs and randomized complete block designs using three types of data. The three types were simulated data with known covariance structure, data from uniformity trials and data from actual trials using incomplete split-plot designs for comparing cereal varieties under different growing conditions. It is concluded that the incomplete split-plot designs may be a good alternative to traditional split-plots and a good compromise between split-plots and randomised complete blocks.     

Predicting spring barley yield from variety-specific yield potential, disease resistance and straw length, and from environment-specific disease loads and weed pressure

For low-input crop production, well-characterised varieties increase the possibilities of managing diseases and weeds. This analysis aims at developing a framework for analyzing grain yield using external varietal information about disease resistance, weed competitiveness and yield potential and quantifying the impact of susceptibility grouping and straw length scores (as a measure for weed competitiveness) for predicting spring barley grain yield under variable biotic stress levels. The study comprised 52 spring barley varieties and 17 environments, i.e., combinations of location, growing system and year. Individual varieties and their interactions with environments were analysed by factorial regression of grain yield on external variety information combined with observed environmental disease loads and weed pressure. The external information was based on the official Danish VCU testing. The most parsimonious models explained about 50% of the yield variation among varieties including genotype-environment interactions. Disease resistance characteristics of varieties, weighted with disease loads of powdery mildew, leaf rust and net blotch, respectively, had a highly significant influence on grain yield. The extend to which increased susceptibility resulted in increased yield losses in environments with high disease loads of the respective diseases was predicted. The effect of externally determined straw length scores, weighted with weed pressure, was weaker although significant for weeds with creeping growth habit. Higher grain yield was thus predicted for taller plants under weed pressure. The results are discussed in relation to the model framework, impact of the considered traits and use of information from conventional variety testing in organic cropping systems.     

Differentiation of species from the Penicillium roqueforti group by volatile metabolite profiling

Species from the Penicillium roqueforti group were differentiated by volatile metabolite profiling primarily of sesquiterpenes. A total of 24 isolates from species P. roqueforti, Penicillium carneum, and the recently described species Penicillium paneum were inoculated on yeast extract sucrose agar. Volatile metabolites were collected by diffusive sampling onto tubes containing Tenax TA, overnight between the fifth and sixth days of incubation. Volatiles were thermally desorbed and analyzed by gas chromatography coupled to mass spectrometry. The sesquiterpene area of the chromatogram was investigated, and potential sesquiterpenes were tabulated by comparison of their Kovats retention index and mass spectrum. In general, P. carneum isolates produced the lowest number of sesquiterpenes, all of which were unique for P. carneum within the P. roqueforti group. P. roqueforti and P. paneum produced a larger variety of volatile metabolites, some of which they have in common and some of which are unique for the two species. (+)-Aristolochene was found in samples from P. paneum and P. roqueforti. Other Penicillium species in which (+)-aristolochene was also detected were P. commune, P. glandicola, and P. solitum.     

Nitrogen effects of non-legume catch crops

Nitrate leaching during the winter period can be reduced and often prevented by growing catch crops after the harvest of a main crop. However, catch crops which effectively take up residual nitrogen do not necessarily show good nutrient effects on a succeeding main crop. The objective of this experiment was to investigate how the content of soil mineral nitrogen in spring was affected by the time of incorporation of non-legume catch crops and how the yield and nitrogen uptake of a succeeding main crop was influenced. The yield of spring sown onion and white cabbage was significantly increased by catch crop growing the previous autumn. The nitrogen effect of Italian ryegrass corresponded to 50–100 kg N per ha in the vegetables. However, the yield of spring barley was not significantly affected by the nitrogen released from decomposing catch crops. During decomposition of non-legume catch crops, grown at a high level of nitrogen fertility, nitrogen immobilization did not occur.