G-quadruplexes are specifically recognized and distinguished by selected designed ankyrin repeat proteins

We introduce designed ankyrin repeat binding proteins (DARPins) as a novel class of highly specific and structure-selective DNA-binding proteins, which can be functionally expressed within all cells. Human telomere quadruplex was used as target to select specific binders with ribosome display. The selected DARPins discriminate the human telomere quadruplex against the telomeric duplex and other quadruplexes. Affinities of the selected binders range from 3 to 100 nM. CD studies confirm that the quadruplex fold is maintained upon binding. The DARPins show different specificity profiles: some discriminate human telomere quadruplexes from other quadruplex-forming sequences like ILPR, c-MYC and c-KIT, while others recognize two of the sequences tested or even all quadruplexes. None of them recognizes dsDNA. Quadruplex-binding DARPins constitute valuable tools for specific detection at very small scales and for the in vivo investigation of quadruplex DNA.     

Optimization of allosteric MEK inhibitors. Part 1: Venturing into underexplored SAR territories

Using PD325901 as a starting point for identifying novel allosteric MEK inhibitors with high cell potency and long-lasting target inhibition in vivo, truncation of its hydroxamic ester headgroup was combined with incorporation of alkyl and aryl ethers at the neighboring ring position. Whereas alkoxy side chains did not yield sufficient levels of cell potency, specifically substituted aryloxy groups allowed for high enzymatic and cellular potencies. Sulfamide 28 was identified as a highly potent MEK inhibitor with nanomolar cell potency against B-RAF (V600E) as well as Ras-mutated cell lines, high metabolic stability and resulting long half-lives. It was efficacious against B-RAF as well as K-Ras driven xenograft models and showed—despite being orally bioavailable and not a P-glycoprotein substrate—much lower brain/plasma exposure ratios than PD325901.     

Although there is no Physical Short‐Term Scarcity of Phosphorus,its Resource Efficiency Should be Improved

The German government has adopted a law that requires sewage plants to go beyond the recovery of phosphorus from wastewater and to promote recycling. We argue that there is no physical global short‐ or mid‐term phosphorus scarcity. However, we also argue that there are legitimate reasons for policies such as those of Germany, including: precaution as a way to ensure future generations’ long‐term supply security, promotion of technologies for closed‐loop economics in a promising stage of technology development, and decrease in the current supply risk with a new resource pool.     

One step cloning of defined DNA fragments from large genomic clones

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.     

Down-regulation of IRAK-4 is a component of LPS- and CpG DNA-induced tolerance in macrophages

Macrophages are important mediators of the immune response to infection by virtue of, amongst other things, their ability to secrete cytokines (e.g. TNF) that trigger inflammation. However, excessive systemic release of inflammatory cytokines can cause septic shock and ultimately death. Tolerance is an adaptive mechanism that prevents macrophage activation and inflammatory cytokine production. The activation of macrophages by pathogens is largely mediated by Toll-like receptors (TLRs). IRAK-4 and IRAK-1 are proximal protein kinases in TLR signalling pathways; IRAK-1 is activated via its phosphorylation by IRAK-4. The rapid degradation of IRAK-1 following its TLR-induced activation has been proposed to represent a major mechanism for tolerance. Here, we established that IRAK-1 degradation is insufficient to cause tolerance; in the absence of IRAK-1, IRAK-4 likely activates downstream signalling proteins (e.g. NF-kappaB) via IRAK-2. Significantly, tolerance coincided with IRAK-4 down-regulation, which occurred at the protein level via proteolytic degradation as well as at the mRNA level. Gene silencing experiments confirmed the importance of IRAK-4 for the regulation of TNF expression. The different kinetics of IRAK-4 and IRAK-1 down-regulation may result in both quantitative and qualitative differences in TLR signalling and potentially allow macrophages to temporally modify their inflammatory responses. Furthermore, differences in the kinetics and extent of IRAK-4 down-regulation by TLR ligands may provide a mechanism whereby macrophages can tailor their inflammatory response according to the location and/or type of pathogen detected.     

Biophysical properties and functional significance of stem water storage tissues in Neotropical savanna trees

Biophysical characteristics of sapwood and outer parenchyma water storage compartments were studied in stems of eight dominant Brazilian Cerrado tree species to assess the impact of differences in tissue capacitance on whole-plant water relations. The rate of decline in tissue water potential with relative water content (RWC) was greater in the outer parenchyma than in the sapwood for most of the species, resulting in tissue-and species-specific differences in capacitance. Sapwood capacitance on a tissue volume basis ranged from 40 to 160 kg m-3 MPa-1, whereas outer parenchyma capacitance ranged from 25 to only 60 kg m-3 MPa-1. In addition, osmotic potentials at full turgor and at the turgor loss point were more negative for the outer parenchyma compared with the sapwood, and the maximum bulk elastic modulus was higher for the outer parenchyma than for the sapwood. Sapwood capacitance decreased linearly with increasing sapwood density across species, but there was no significant correlation between outer parenchyma capacitance and tissue density. Midday leaf water potential, the total hydraulic conductance of the soil/leaf pathway and stomatal conductance to water vapour (gs) all increased with stem volumetric capacitance, or with the relative contribution of stored water to total daily transpiration. However, the difference between the pre-dawn water potential of non-transpiring leaves and the weighted average soil water potential, a measure of the water potential disequilibrium between the plant and soil, increased asymptotically with total stem capacitance across species, implying that overnight recharge of water storage compartments was incomplete in species with greater capacitance. Overall, stem capacitance contributes to homeostasis in the diurnal and seasonal water balance of Cerrado trees.     

Tet repressor induction without Mg2+

We have examined anhydrotetracycline (atc) binding to Tet repressor (TetR) in dependence of the Mg(2+) concentration. Of all tc compounds tested so far, atc has the highest affinity for TetR, with a K(A) of 9.8 x 10(11) M(-1) in the presence of Mg(2+) and 6.5 x 10(7) M(-1) without Mg(2+). Thus, it binds TetR with 500-fold higher affinity than tc under both conditions. The Mg(2+)-free binding of atc to TetR leads to induction in vitro, demonstrating that the metal is not necessary to trigger the associated conformational change. To obtain more detailed information about Mg(2+)-free induction, we constructed and prepared to homogeneity four single-alanine substitution mutants of TetR. Three of them affect residues involved in contacting Mg(2+) (TetR H100A, E147A, and T103A), and one altered residue contacts tc TetR N82A. TetR H100A and E147A are induced by atc, with and without Mg(2+), showing 110-fold and 1000-fold decreased Mg(2+)-dependent and unchanged Mg(2+)-independent atc binding, respectively. Thus, the contacts of these residues to Mg(2+) are not necessary for induction. TetR N82A is not inducible under any of the conditions employed and shows an about 4000-fold decreased atc binding constant. The Mg(2+)-dependent affinity of TetR T103A for atc is only 400-fold reduced, but no induction with atc was observed. Thus, Thr103 must be essential for the conformational change associated with induction in the absence of Mg(2+).     

Constant morphological patterns in the hemolymph vascular system of crayfish (Crustacea,Decapoda)

We present a study of the hemolymph vascular system of the marbled crayfish, Procambarus fallax f. virginalis, the only crayfish species known to be parthenogenetic. To identify potential evolutionary patterns, we compared data from a total of 48 specimens of P. fallax with 22 specimens of Orconectes limosus. Visualizations (2D and 3D) were carried out using a combination of classical and modern morphological techniques. Our data were compared to the existing literature.Like all Decapoda, both P. fallax and O. limosus have a hemolymph vascular system, consisting of a globular heart with seven off-branching arteries. We were able to visualize in detail the heart of crayfish for the first time, i.e., the myocard with its clusters of muscles running through the lumen of the heart, the valves and flaps of ostia and arteries. Furthermore, the branching patterns of the seven artery systems were analyzed. Anatomical structures identified to be consistent in all specimens of both species were combined as ground pattern of hemolymph vascular system features for Astacida.     

Measuring Preferences on Environmental Damages in LCIA. Part 1: Cognitive Limits in Panel Surveys (9 pp)

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Part 1: Cognitive Limits in Panel Surveys · Part 2: The Question Format in Panel Surveys This series of two papers discusses the elicitation of weights for damage categories in LCIA with the aid of panel surveys. The papers focus especially on methodological aspects in panel surveys. Part 1 discusses potential cognitive limits of the panel members to understand the reference that is weighted. Part 2 focuses on the influence of the question format and compares results from two different weighting tasks: discrete choice (between alternatives) and score allocation.

Goal, Scope and Background

The weighting of environmental impacts and damages on the safeguard subjects Human Health, Ecosystems, and Resources is a significant step of full aggregated LCIA. Panel surveys have become a common approach in LCIA research to investigate the preferences of stakeholders on environmental impacts and damages. Despite the numerous studies, the knowledge on how to elicit reliable weights is still poor and inconsistent. We present a questionnaire study with 58 environmental science students to investigate so-called framing effects in panel surveys.

Main Features

The study investigates the significance of different framings, which were provided by three references. In addition, the significance of quantitative information provided in the questionnaire is tested. The references are (a1) safeguard subjects without specified additional information, (a2) damages in Europe as they are perceived by the panelist, and (a3) quantified scenarios derived from Eco-indicator99. All participants ranked and rated the importance of the safeguard subjects three times, once within each reference system. According to a test-of-scope study, quantitative information given to the panelist was varied. One level (b1) included data from the Ecoindicator99 methodology, whereas the other group (b2) received data with significantly higher Human Health damages and lower Ecosystem damages, ceteris paribus. This design allows testing the influence of quantitative data on the rating.

Results

The weighting of the safeguard subjects (a1) reveals that Human Health is considered a slightly more important safeguard subject than Ecosystems. However, both are judged to be significantly more important than Resources. This picture changes for the references (a2) and (a3) where damages were weighted. For both references, the respondents rated damages to Ecosystems as most important followed by Resources and Human Health, showing by far the lowest weights. Therefore, the framing of the reference that was weighted played a significant role. The ratings of the subgroups (b1) and (b2) did not differ with respect to the importance of damages, though substantially different quantitative information was given.

Conclusion and Outlook

The participants of the study were obviously insensitive with respect to quantitative information provided. This raises three questions, which are discussed. What is the mental model upon which respondents base their beliefs and values? Can we expect that 'more sophisticated' subjects would respond differently? Which prerequisites should an empirical weighting procedure fulfill in order to incorporate numerical data? We propose different approaches for future procedures in order to accurately analyze these questions.