NMR-based metabonomics of bovine blood: an investigation into the effects of long term storage on plasma samples

Freezers in research institutions often contain a plethora of samples left over from studies performed years or even decades ago. Along with samples stored in biobanks, these could prove to be treasure troves for metabonomic research. Although the influence of sample handling and short to medium term storage on conventionally determined blood parameters has been reported, little is known about the effects of long term storage (years to decades) on plasma samples. The aim of this study was to investigate the influence of long term storage on the metabolite profile and to assess the value of archived samples for metabonomic studies. Heparinised plasma of 22 heifers that had been stored at ?20 °C for between 2 and 15 years was analysed using NMR spectroscopy and statistical analysis techniques. Lactate (principal component 1) explained 79.6 % of variance between all spectra, but was not correlated with storage time. The highest correlation with storage time (R ² = 0.474) was found for betaine, with other metabolites (acetoacetate, histidines, glycerol, lipids and glucose) also showing moderate correlation (R ² values between 0.217 and 0.437). Our results indicate that samples stored for extended periods of time can potentially be used in metabonomics studies, if precautions are taken during data analysis.     

Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.     

Surface active adrenoceptor compounds prevent the settlement of cyprid larvae of Balanus improvisus

The barnacle Balanus improvisus is the major fouling macroorganism in Swedish waters and it colonizes most man‐made surfaces submerged in the sea. New or impending legislation restricts the use of traditional, hazardous antifouling coatings based on heavy metals, mainly copper and tin. This calls for the development of new non‐toxic methods that prevent barnacle settlement. In this work several adrenoceptor compounds are shown to be very efficient in preventing the settlement of cyprid larvae of B. improvisus. The settlement rate of laboratory‐reared cyprids was studied in hydrophilised polystyrene dishes containing adrenoceptor antagonists and agonists dissolved in seawater. Two of these drugs, medetomidine and clonidine, repeatedly inhibited settlement at concentrations between 1 nM and 10 nM. In the vertebrate adrenoceptor classification system, which separates pharmacological substances according to their receptor affinity, both of these substances are classified as α₂ adrenoceptor agonists. An inhibiting effect on presyn‐aptic receptors is suggested, but the localization of the receptor effect requires futher studies. Experiments also revealed that the inhibiting effect of medetomidine was reversible. Cyprids incubated with medetomidine for 20 h attached and metamorphosed into juvenile barnacles after washing and transferrence to seawater. The antagonizing compound atipamezole reversed the effect of medetomidine. This observation supports the assumption that this substance acts at the receptor level. Studies of the surface affinity of medetomidine revealed a strong tendency to accumulate in solid/ liquid phase boundaries. This ability makes it particularly attractive as a candidate for the development of a slow‐release carrier in marine coatings. Panels coated with medetomidine in an acrylate polymer and exposed in the field reduced the recruitment of B. improvisus by 96% after 4 weeks and by 70% after 8 weeks.     

Oxygen-depleted surfaces: a new antifouling technology

A novel, non-toxic strategy to combat marine biofouling is presented. The technology is paint with additions of up to 43% of industrial protein. Through microbial degradation of the protein component, an oxygen-depleted layer rapidly forms in a 0.2 mm layer close to the paint surface. With the present paint formulations, a stable, O₂-depleted layer can persist for 16 weeks. Barnacle larvae (cyprids) did not settle on panels where oxygen saturation was <20%, and cyprids were killed when exposed to O₂-free water for more than 1 h. It is also shown that the O₂-depleted layer will rapidly reform (within 15 min) after exposure to turbulent flow. Field exposure of panels for 16 weeks showed that paint with protein reduced fouling by barnacles and bryozoans by 80% and close to 100%, respectively. The results suggest that this novel technology may be developed into a non-toxic alternative to copper-based antifouling paints, especially for pleasure boats in sensitive environments. There is clearly potential for further development of the paint formulation, and a full-scale test on a boat-hull suggested that service-life under realistic operations needs to be improved.     

A role for oxalic acid generation in ozone‐induced signallization in Arabidopis cells

Ozone (O₃) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O₃ results in ascorbate‐derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca²⁺ concentration, generation of reactive oxygen species and cell death. We confirmed that O₃ reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O₃‐induced signalling pathways that trigger programmed cell death.     

PINT: a software for integration of peak volumes and extraction of relaxation rates

We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.     

Resilience of ecosystem processes: a new approach shows that functional redundancy of biological control services is reduced by landscape simplification

Functional redundancy can increase the resilience of ecosystem processes by providing insurance against species loss and the effects of abundance fluctuations. However, due to the difficulty of assessing individual species’ contributions and the lack of a metric allowing for a quantification of redundancy within communities, few attempts have been made to estimate redundancy for individual ecosystem processes. We present a new method linking interaction metrics with metabolic theory that allows for a quantification of redundancy at the level of ecosystem processes. Using this approach, redundancy in the predation on aphids and other prey by natural enemies across a landscape heterogeneity gradient was estimated. Functional redundancy of predators was high in heterogeneous landscapes, low in homogeneous landscapes and scaled with predator specialisation. Our approach allows quantifying functional redundancy within communities and can be used to assess the role of functional redundancy across a wide variety of ecosystem processes and environmental factors.     

Imaging of disease dynamics during meningococcal sepsis

Neisseria meningitidis is a human pathogen that causes septicemia and meningitis with high mortality. The disease progression is rapid and much remains unknown about the disease process. The understanding of disease development is crucial for development of novel therapeutic strategies and vaccines against meningococcal disease. The use of bioluminescent imaging combined with a mouse disease model allowed us to investigate the progression of meningococcal sepsis over time. Injection of bacteria in blood demonstrated waves of bacterial clearance and growth, which selected for Opa-expressing bacteria, indicating the importance of this bacterial protein. Further, N. meningitidis accumulated in the thyroid gland, while thyroid hormone T4 levels decreased. Bacteria reached the mucosal surfaces of the upper respiratory tract, which required expression of the meningococcal PilC1 adhesin. Surprisingly, PilC1 was dispensable for meningococcal growth in blood and for crossing of the blood-brain barrier, indicating that the major role of PilC1 is to interact with mucosal surfaces. This in vivo study reveals disease dynamics and organ targeting during meningococcal disease and presents a potent tool for further investigations of meningococcal pathogenesis and vaccines in vivo. This might lead to development of new strategies to improve the outcome of meningococcal disease in human patients.     

Long‐term rearing affects pheromone‐mediated flight behaviour of the Indian meal moth,Plodia interpunctella

The pest Plodia interpunctella (Hübner) is reared in many research laboratories. In a culture established in 1996, attraction of males to the female‐produced sex pheromone in flight tunnel assays gradually decreased after ≈15 years of rearing. A new culture was established to enable comparison with the old culture regarding traits associated with mate finding. Female calling activity, pheromone titre and male antennal response to pheromone components did not differ between cultures. In contrast, very few males from the old culture reached the pheromone source in flight tunnel assays compared with 61%–81% of males from the other culture. Our results highlight the importance of maintaining viable insect cultures for research purposes and suggest frequent evaluation of traits involved in chemical communication in such cultures to ensure reliable results in experiments.     

Are certain life histories particularly prone to local extinction?

Using stochastic simulations and elasticity analysis, we show that there are inherent differences in the risk of extinction between life histories with different demographies. Which life history is the most vulnerable depends on which vital rate varies. When juvenile survival varies semelparous organisms with delayed reproduction are the most vulnerable ones, while a varying developmental rate puts a greater threat to semelparous organisms with rapid development. Iteroparous organisms are the most vulnerable ones when adult survival varies. Generally, we do not expect to observe organisms in nature having variation in vital rates that produce a high risk of extinction. Given the results here we therefore predict that iteroparous organisms should show low variation in adult survival. Moreover, we predict that semelparous organisms should show low variation in juvenile survival and low variation in developmental rate. The effect of temporal correlation on extinction risk is most pronounced in organisms with semelparous life histories.