Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.The evolving maturation of the field of proteomics has, in the same way as in genomics, highlighted the need of better sampling procedures and sample preparation methodologies to minimize the effect of post mortem alterations. The aspect of sample quality is not new in any way and is relevant in most biomedical fields but has only lately started to receive adequate attention. The main factors influencing sample quality is storage temperature of the body until tissue removal (foremost a problem in clinical settings and extraction of less accessible tissue samples from model organisms) and post mortem interval (PMI)¹ (1–3). Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2, 3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3–8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1, 3–12). There are also a few proteomic studies on muscle tissue degradation in livestock (13–16).We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to post-translational modification (PTM) stability (2, 3, 17–20). An alternative method based on cryostat dissection with subsequent heat treatment through boiling has also been reported to improve endogenous peptide sample quality (21). Besides focused microwave irradiation, which is specifically used for rodent brain tissue sampling, we have also demonstrated the efficiency of rapid heat stabilization through conductivity with regard to sample degradation (3, 22). Although somewhat constrained by its dependence on how quickly the tissue is harvested from the body, the latter procedure has the added advantage that it can be used on any type of tissue and species, fresh as well as frozen. This study will compare effects of sampling procedures on the liver and pancreas degradome following rapid heat stabilization, the more traditional snap freezing, or the combination of snap freezing with subsequent heat stabilization.To summarize, this study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display (2, 18), the later to better investigate changes in small molecular fragment that are not easily detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few minutes PMI (3). The peptidomics approach has been used in several studies to point out early post mortem changes and protein degradation that tissue undergo following sampling and is therefore a well-suited method (3, 18, 22).     

Species-Rich Scandinavian Grasslands are Inherently Open to Invasion

Invasion of native habitats by alien or generalist species is recognized worldwide as one of the major causes behind species decline and extinction. One mechanism determining community invasibility, i.e. the susceptibility of a community to invasion, which has been supported by recent experimental studies, is species richness and functional diversity acting as barriers to invasion. We used Scandinavian semi-natural grasslands, exceptionally species-rich at small spatial scales, to examine this mechanism, using three grassland generalists and one alien species as experimental invaders. Removal of two putative functional groups, legumes and dominant non-legume forbs, had no effect on invasibility except a marginally insignificant effect of non-legume forb removal. The amount of removed biomass and original plot species richness had no effect on invasibility. Actually, invasibility was high already in the unmanipulated community, leading us to further examine the relationship between invasion and propagule pressure, i.e. the inflow of seeds into the community. Results from an additional experiment suggested that these species-rich grasslands are effectively open to invasion and that diversity may be immigration driven. Thus, species richness is no barrier to invasion. The high species diversity is probably in itself a result of the community being highly invasible, and species have accumulated at small scales during centuries of grassland management.     

Morningness: protective factor for sleep-related and emotional problems in childhood and adolescence?

The relationship between morningness/eveningness, sleep, and psychological problems is well documented in adults as well as in adolescents. However, research on the circadian orientation and its concomitants in younger children is scarce. The authors investigated the distribution of morningness/eveningness and its connection to sleeping and psychological problems in 91 children and 151 adolescents in Austria. The authors found that morning (M) types had less sleep-related and psychological problems than intermediate (I) and evening (E) types, respectively. Among children, M-types suffered less from daytime sleepiness (females: χ(2)((2))?= 8.1, p =?.017; males: χ(2)((2))?= 14.8, p =?.001). Among adolescents, M-types showed fewer sleep-wake problems (females: χ(2)((2))?= 17.5, p 相似文献   

Fostering responsible research with genome editing technologies: a European perspective

In this consensus paper resulting from a meeting that involved representatives from more than 20 European partners, we recommend the foundation of an expert group (European Steering Committee) to assess the potential benefits and draw-backs of genome editing (off-targets, mosaicisms, etc.), and to design risk matrices and scenarios for a responsible use of this promising technology. In addition, this European steering committee will contribute in promoting an open debate on societal aspects prior to a translation into national and international legislation.     

Inhibition of Mitochondrial Complex III Blocks Neuronal Differentiation and Maintains Embryonic Stem Cell Pluripotency

The mitochondrion is emerging as a key organelle in stem cell biology, acting as a regulator of stem cell pluripotency and differentiation. In this study we sought to understand the effect of mitochondrial complex III inhibition during neuronal differentiation of mouse embryonic stem cells. When exposed to antimycin A, a specific complex III inhibitor, embryonic stem cells failed to differentiate into dopaminergic neurons, maintaining high Oct4 levels even when subjected to a specific differentiation protocol. Mitochondrial inhibition affected distinct populations of cells present in culture, inducing cell loss in differentiated cells, but not inducing apoptosis in mouse embryonic stem cells. A reduction in overall proliferation rate was observed, corresponding to a slight arrest in S phase. Moreover, antimycin A treatment induced a consistent increase in HIF-1α protein levels. The present work demonstrates that mitochondrial metabolism is critical for neuronal differentiation and emphasizes that modulation of mitochondrial functions through pharmacological approaches can be useful in the context of controlling stem cell maintenance/differentiation.     

Studying hunting behaviour in the striped field mouse using data compression

We compare predatory behaviour towards a mobile insect in three species of small mammals: the granivorous striped field mouse, the insectivorous common shrew and the Norway rat (a generalist). The striped field mouse displays a surprisingly efficient hunting stereotype. We apply the data compression method (Ryabko et al. Theory Comput Syst 52:133–147, 2013) to compare the complexity of hunting behavioural patterns and to evaluate the flexibility of stereotypes and their succinctness. Norway rats demonstrated the highest level of complexity of hunting behaviour, with the highest proportion of ‘auxiliary’ and ‘noise’ elements and relatively low proportion of ‘key’ elements in their behaviours. The predominance of ‘key’ elements resulted in similarly low levels of complexity of hunting stereotypes in striped field mice and shrews. The similarity between hunting stereotypes of the insectivorous shrew and the granivorous striped field mouse enables us to argue about evolutionary roots of hunting behaviour in small mammals. We show that this method is a useful tool for comparing ethograms as ‘biological texts’.     

Development of foraging skills in two orangutan populations: needing to learn or needing to grow?

Background

Orangutans have one of the slowest-paced life histories of all mammals. Whereas life-history theory suggests that the time to reach adulthood is constrained by the time needed to reach adult body size, the needing-to-learn hypothesis instead suggests that it is limited by the time needed to acquire adult-level skills.To test between these two hypotheses, we compared the development of foraging skills and growth trajectories of immature wild orangutans in two populations: at Tuanan (Pongo pygmaeus wurmbii), Borneo, and Suaq Balimbing (Pongo abelii), Sumatra. We collected behavioral data on diet repertoire, feeding rates and ranging competence during focal follows, and estimated growth through non-invasive laser photogrammetry.

Results

We found that adult-like diet repertoires are attained around the age of weaning and that female immatures increase their repertoire size faster than their male peers. Adult-level feeding rates of easy techniques are reached just after weaning, but several years later for more difficult techniques, albeit always before adulthood (i.e. age at first reproduction). Independent immatures had faster feeding rates for easy to process items than their mothers, with male immatures achieving faster feeding rates earlier in development relative to females. Sumatran immatures reach adult-level feeding rates 2–3 years later than their Bornean peers, in line with their higher dietary complexity and later weaning. The range-use competence of independently ranging and weaned immatures is similar to that of adult females. Body size measurements showed, immatures grow until female age of first reproduction.

Conclusions

In conclusion, unlike in humans, orangutan foraging skills are in place prior to reproduction. Growth trajectories suggest that energetic constraints, rather than skills, best explain the length of immaturity. However, skill competence for dietary independence is reached later where the adult niche is more complex, which is consistent with the relatively later weaning age with increasing brain size found generally in primates, and apes in particular.

     

Evidence for rapid evolution in a grassland biodiversity experiment

In long‐term grassland experiments, positive biodiversity effects on plant productivity commonly increase with time. Subsequent glasshouse experiments showed that these strengthened positive biodiversity effects persist not only in the local environment but also when plants are transferred into a common environment. Thus, we hypothesized that community diversity had acted as a selective agent, resulting in the emergence of plant monoculture and mixture types with differing genetic composition. To test our hypothesis, we grew offspring from plants that were grown for eleven years in monoculture or mixture environments in a biodiversity experiment (Jena Experiment) under controlled glasshouse conditions in monocultures or two‐species mixtures. We used epiGBS, a genotyping‐by‐sequencing approach combined with bisulphite conversion, to provide integrative genetic and epigenetic (i.e., DNA methylation) data. We observed significant divergence in genetic and DNA methylation data according to selection history in three out of five perennial grassland species, namely Galium mollugo, Prunella vulgaris and Veronica chamaedrys, with DNA methylation differences mostly reflecting the genetic differences. In addition, current diversity levels in the glasshouse had weak effects on epigenetic variation. However, given the limited genome coverage of the reference‐free bisulphite method epiGBS, it remains unclear how much of the differences in DNA methylation was independent of underlying genetic differences. Our results thus suggest that selection of genetic variants, and possibly epigenetic variants, caused the rapid emergence of monoculture and mixture types within plant species in the Jena Experiment.