Lars-Henrik Heckmann

NORMA-Gene: a simple and robust method for qPCR normalization based on target gene data.

BackgroundNormalization of target gene expression, measured by real-time quantitative PCR (qPCR), is a requirement for reducing experimental bias and thereby improving data quality. The currently used normalization approach is based on using one or more reference genes. Yet, this approach extends the experimental work load and suffers from assumptions that may be difficult to meet and to validate.ResultsWe developed a data driven normalization algorithm (NORMA-Gene). An analysis of the performance of NORMA-Gene compared to reference gene normalization on artificially generated data-sets showed that the NORMA-Gene normalization yielded more precise results under a large range of parameters tested. Furthermore, when tested on three very different real qPCR data-sets NORMA-Gene was shown to be best at reducing variance due to experimental bias in all three data-sets compared to normalization based on the use of reference gene(s).ConclusionsHere we present the NORMA-Gene algorithm that is applicable to all biological and biomedical qPCR studies, especially those that are based on a limited number of assayed genes. The method is based on a data-driven normalization and is useful for as little as five target genes comprising the data-set. NORMA-Gene does not require the identification and validation of reference genes allowing researchers to focus their efforts on studying target genes of biological relevance.

Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.

Macroinvertebrate community response to pulse exposure with the insecticide lambda-cyhalothrin using in-stream mesocosms

Pesticides are constantly being applied to agricultural catchments, but little is known about their impact on aquatic biota during natural exposure. In the present study, the impact of the pyrethroid lambda-cyhalothrin was studied in an in-stream mesocosm setup. Twice during the summer of 2002, the natural macroinvertebrate community was exposed in situ to a 30-min pulse of lambda-cyhalothrin. Pyrethroid doses were released through a modified drip set with nominal concentrations of 0.10, 1.00, and 10.0 microg L(-1) during the first exposure and 0.05, 0.50, and 5.00 microg L(-1) in the second exposure. Before, during, and after exposure, drifting macroinvertebrates were caught in nets. Quantitative benthic samples were taken both before and on two occasions after exposure. Macroinvertebrate drift increased immediately after the pulse exposure, with total drift being significantly higher at all concentrations. Gammarus pulex, various Ephemeroptera, Leuctra sp., and Simuliidae were some of the taxa showing the most pronounced drift response. Structural change in the community was found only at 5.00 and 10.0 microg L(-1), and recovery occurred within approximately two weeks. The present study may be valuable in assessing extrapolations based on laboratory results as well as in evaluating pyrethroid impact on natural freshwater environments.

Time-course profiling of molecular stress responses to silver nanoparticles in the earthworm Eisenia fetida.

The molecular mechanism of silver nanoparticle (AgNP) toxicity, particularly its temporal aspect, is currently limited in the literature. This study seeks to identify and profile changes in molecular response patterns over time during soil exposure of the earthworm Eisenia fetida to AgNPs (82±27 nm) with reference to dissolved silver salt (AgNO₃). Principal component analysis of selected gene and enzyme response profiles revealed dissimilar patterns between AgNO₃ and AgNP treatments and also over time. Despite the observed difference in molecular profiles, the body burdens of total Ag were within the same range (10-40 mg/kg dry weight worm) for both treatments with apparent correlation to the induction pattern of metallothionein. AgNO₃ induced the genes and enzymes related to oxidative stress at day 1, after which markers of energy metabolism were all suppressed at day 2. Exposure to AgNPs likewise led to induction of oxidative stress genes at day 2, but with a temporal pattern shift to immune genes at day 14 following metabolic upregulation at day 7. The involvement of oxidative stress and subsequent alterations in immune gene regulation were as predicted by our in vitro study reported previously, highlighting the importance of immunological endpoints in nanosilver toxicity.

Time is of the essence

An organism is a dynamic system, and its life history results from underlying processes in time. The effects of biological and chemical stressors on this system therefore also change temporally. In the present short communication, we emphasize the importance of including time as a factor in stress ecology and ecotoxicology and argue that current standard test protocols may benefit considerably from this, improving data interpretation and thus also risk assessment and risk management.

Hsp70 expression and metabolite composition in response to short-term thermal changes in Folsomia candida (Collembola)

In the present study the joint transcriptomic and metabolomic responses in Folsomia candida (Collembola) to temperature changes on a short-term scale were studied. Change in heat tolerance was examined as survival after a 35 degrees C heat shock (2h) in the course of either a fluctuating temperature regime (8 to 32 degrees C; pre-treated) or a constant temperature (20 degrees C; control) over a period of 24h. Exposure to a temperature increase from 20 to 32 degrees C (2.4 degrees C min(-)(1)) induced a significantly increased heat tolerance which continued throughout the experiment. Expression of the gene encoding heat shock protein Hsp70 was assessed at the mRNA level using real time quantitative polymerase chain reaction (QPCR). Hsp70 was rapidly induced and significantly increased by the temperature increase. The relative concentrations of low molecular weight metabolites were analysed in F. candida using nuclear magnetic resonance spectroscopy ((1)H NMR). A significant metabolomic divergence between pre-treated and control collembolans was evident; partly due to a significantly reduced relative concentration of five free amino acids (arginine, leucine, lysine, phenylalanine and tyrosine) in pre-treated collembolans. These results, obtained from ecological transcriptomics and metabolomics jointly generated insight on various levels into the combined responses to a changing environment.

Exposure to mercury reduces heat tolerance and heat hardening ability of the springtail Folsomia candida

We investigated the combined effects of mercury (HgCl(2)) and acute heat on survival of the springtail Folsomia candida. The springtails were exposed to a range of aqueous concentrations (0-48 mg Hg(2+)/L) of HgCl(2) for 24 h. Subsequently, the same individuals were exposed to a range of high temperatures, from 20 to 35.5 degrees C. We found a highly significant synergistic interaction between effects of mercury and heat, with a reduced tolerance to heat after exposure to sublethal concentrations of mercury. Further, the heat hardening ability of F. candida was studied at sublethal concentrations of mercury. F. candida was able to heat harden (exposure to a mild heat treatment increasing survival of subsequent severe heat); however, when the springtails experienced a previous exposure to as little as 1 mg Hg(2+)/L, heat hardening failed to improve survival of heat shock at 34.5 degrees C, even though this was much lower than concentrations affecting survival without heat stress. Mild heat stress is known to induce the heat shock protein, HSP70, and real-time quantitative PCR confirmed that pre-acclimation to 32 degrees C did indeed cause >5-fold up-regulation of HSP70 expression. This up-regulation was not affected by previous exposure to 1 mg Hg(2+)/L.

Insects as food and feed: from production to consumption

Alternative protein sources are urgently required as the available land area is not sufficient to satisfy the growing demand for meat. Insects have a high potential of becoming a new sector in the food and feed industry, mainly because of the many environmental benefits when compared to meat production. This will be outlined in the book, as well as the whole process from rearing to marketing. The rearing involves large scale and small scale production, facility design, the management of diseases , and how to assure that the insects will be of high quality (genetics). The nutrient content of insects will be discussed and how this is influenced by life stage, diet, the environment and processing. Technological processing requires decontamination, preservation, and ensuring microbial safety. The prevention of health risks (e.g. allergies) will be discussed as well as labelling, certification and legislative frameworks. Additional issues are: insect welfare, the creation of an enabling environment, how to deal with consumers, gastronomy and marketing strategies. Examples of production systems will be given both from the tropics and from temperate zones.