Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of K_d values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.     

Genotype-based databases for variants causing rare diseases

Inherited diseases are the result of DNA sequence changes. In recessive diseases, the clinical phenotype results from the combined functional effects of variants in both copies of the gene. In some diseases there is often considerable variability of clinical presentation or disease severity, which may be predicted by the genotype. Additional effects may be triggered by environmental factors, as well as genetic modifiers which could be nucleotide polymorphisms in related genes, e.g. maternal ApoE or ABCA1 genotypes which may have an influence on the phenotype of SLOS individuals. Here we report the establishment of genotype variation databases for various rare diseases which provide individual clinical phenotypes associated with genotypes and include data about possible genetic modifiers. These databases aim to be an easy public access to information on rare and private variants with clinical data, which will facilitate the interpretation of genetic variants.     

Cell Differentiation and Development in Arabidopsis Are Associated with Changes in Histone Dynamics at the Single-Cell Level

The mechanism whereby the same genome can give rise to different cell types with different gene expression profiles is a fundamental problem in biology. Chromatin organization and dynamics have been shown to vary with altered gene expression in different cultured animal cell types, but there is little evidence yet from whole organisms linking chromatin dynamics with development. Here, we used both fluorescence recovery after photobleaching and two-photon photoactivation to show that in stem cells from Arabidopsis thaliana roots the mobility of the core histone H2B, as judged by exchange dynamics, is lower than in the surrounding cells of the meristem. However, as cells progress from meristematic to fully differentiated, core histones again become less mobile and more strongly bound to chromatin. We show that these transitions are largely mediated by changes in histone acetylation. We further show that altering histone acetylation levels, either in a mutant or by drug treatment, alters both the histone mobility and markers of development and differentiation. We propose that plant stem cells have relatively inactive chromatin, but they keep the potential to divide and differentiate into more dynamic states, and that these states are at least in part determined by histone acetylation levels.     

LCA of land-based freight transportation: facilitating practical application and including accidents in LCIA

Purpose

A major task concerning the greening of freight transportation is to influence the process of choosing an appropriate transport solution for a shipment. This paper presents the results of a detailed environmental benchmark study of freight transport chains recorded during a shipper survey administered in Switzerland in 2008.

Materials and methods

For the environmental evaluation, life cycle assessment was applied and enhanced with a new method for integrating damage to human health caused by traffic accidents based on the disability adjusted life year concept.

Results and discussion

The results show that in land-based transport, road generally has a lower environmental performance compared to intermodal and rail-only transport. Exceptions exist, e.g. for long pre- and post-haulage distances in intermodal transport or for very low train-load factors. The most relevant environmental interventions to pay attention to are, according to the methods applied, emissions of CO₂, NO_x and particulates as well as accident damages.

Conclusions

Rail transport is often, but not always, environmentally preferable than truck transport. Accident damages to human health should be included in each benchmark study. For practical application, a simplified benchmark methodology is proposed requiring a reduced level of detail for the input data.     

Hfq reduces envelope stress by controlling expression of envelope‐localized proteins and protein complexes in enteropathogenic Escherichia coli

Gram‐negative bacteria possess several envelope stress responses that detect and respond to damage to this critical cellular compartment. The σ^E envelope stress response senses the misfolding of outer membrane proteins (OMPs), while the Cpx two‐component system is believed to detect the misfolding of periplasmic and inner membrane proteins. Recent studies in several Gram‐negative organisms found that deletion of hfq, encoding a small RNA chaperone protein, activates the σ^E envelope stress response. In this study, we assessed the effects of deleting hfq upon activity of the σ^E and Cpx responses in non‐pathogenic and enteropathogenic (EPEC) strains of Escherichia coli. We found that the σ^E response was activated in Δhfq mutants of all E. coli strains tested, resulting from the misregulation of OMPs. The Cpx response was activated by loss of hfq in EPEC, but not in E. coli K‐12. Cpx pathway activation resulted in part from overexpression of the bundle‐forming pilus (BFP) in EPEC Δhfq. We found that Hfq repressed expression of the BFP via PerA, a master regulator of virulence in EPEC. This study shows that Hfq has a more extensive role in regulating the expression of envelope proteins and horizontally acquired virulence genes in E. coli than previously recognized.     

Improved appreciation of the functioning and importance of biological soil crusts in Europe: the Soil Crust International Project (SCIN)

Here we report details of the European research initiative “Soil Crust International” (SCIN) focusing on the biodiversity of biological soil crusts (BSC, composed of bacteria, algae, lichens, and bryophytes) and on functional aspects in their specific environment. Known as the so-called “colored soil lichen community” (Bunte Erdflechtengesellschaft), these BSCs occur all over Europe, extending into subtropical and arid regions. Our goal is to study the uniqueness of these BSCs on the regional scale and investigate how this community can cope with large macroclimatic differences. One of the major aims of this project is to develop biodiversity conservation and sustainable management strategies for European BSCs. To achieve this, we established a latitudinal transect from the Great Alvar of Öland, Sweden in the north over Gössenheim, Central Germany and Hochtor in the Hohe Tauern National Park, Austria down to the badlands of Tabernas, Spain in the south. The transect stretches over 20° latitude and 2,300 m in altitude, including natural (Hochtor, Tabernas) and semi-natural sites that require maintenance such as by grazing activities (Öland, Gössenheim). At all four sites BSC coverage exceeded 30 % of the referring landscape, with the alpine site (Hochtor) reaching the highest cyanobacterial cover and the two semi-natural sites (Öland, Gössenheim) the highest bryophyte cover. Although BSCs of the four European sites share a common set of bacteria, algae (including cyanobacteria) lichens and bryophytes, first results indicate not only climate specific additions of species, but also genetic/phenotypic uniqueness of species between the four sites. While macroclimatic conditions are rather different, microclimatic conditions and partly soil properties seem fairly homogeneous between the four sites, with the exception of water availability. Continuous activity monitoring of photosystem II revealed the BSCs of the Spanish site as the least active in terms of photosynthetic active periods.     

Automated Protein Turnover Calculations from 15N Partial Metabolic Labeling LC/MS Shotgun Proteomics Data

Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent protein turnover calculations enable the analysis of gigabytes of data within minutes, a prerequisite for systems biology high throughput studies. Here we present a fully automated method for protein turnover calculations from shotgun proteomics data. The approach enables the analysis of complex shotgun LC/MS ¹⁵N partial metabolic labeling experiments. Spectral envelopes of 1419 peptides can be extracted within an hour. The method quantifies turnover by calculating the Relative Isotope Abundance (RIA), which is defined as the ratio between the intensity sum of all heavy (¹⁵N) to the intensity sum of all light (¹⁴N) and heavy peaks. To facilitate this process, we have developed a computer program based on our method, which is freely available to download at http://promex.pph.univie.ac.at/protover.