Improving bioorthogonal protein ubiquitylation by click reaction

Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.     

The architecture of Norway spruce ectomycorrhizae: three-dimensional models of cortical cells, fungal biomass, and interface for potential nutrient exchange

Gathering realistic data on actual fungal biomass in ectomycorrhized fine root systems is still a matter of concern. Thus far, observations on architecture of ectomycorrhizae (ECMs) have been limited to analyses of two-dimensional (2-D) images of tissue sections. This unavoidably causes stereometrical problems that lead to inadequate assumptions about actual size of cells and their arrangement within ECM's functional compartments. Based on extensive morphological investigations of field samples, we modeled the architectural components of an average-sized Norway spruce ECM. In addition to our comprehensive and detailed quantitative data on cell sizes, we studied actual shape and size, in vivo arrangement, and potential nutrient exchange area of plant cortical cells (CCs) using computer-aided three-dimensional (3-D) reconstructions based on semithin serial sections. We extrapolated a factual fungal biomass in ECMs (Hartig net (HN) included) of 1.71 t?ha^?1 FW (0.36 t?ha^?1 DW) for the top 5 cm of soil for an autochthonous, montane, optimum Norway spruce stand in the Tyrolean Alps. The corresponding potential nutrient exchange area in ECMs including main axes of ECM systems, which is defined as the sum of interfaces between plant CCs and the HN, amounts to at least 3.2?×?10⁵?m²?ha^?1. This is the first study that determines the contribution of the HN to the total fungal biomass in ECMs as well as the quantification of its contact area. Our results may stimulate future research on fungal below-ground processes and their impact on the global carbon cycle.     

Exploration of swapping enzymatic function between two proteins: A simulation study of chorismate mutase and isochorismate pyruvate lyase

The enzyme chorismate mutase EcCM from Escherichia coli catalyzes one of the few pericyclic reactions in biology, the transformation of chorismate to prephenate. The isochorismate pyruvate lyase PchB from Pseudomonas aeroginosa catalyzes another pericyclic reaction, the isochorismate to salicylate transformation. Interestingly, PchB possesses weak chorismate mutase activity as well thus being able to catalyze two distinct pericyclic reactions in a single active site. EcCM and PchB possess very similar folds, despite their low sequence identity. Using molecular dynamics simulations of four combinations of the two enzymes (EcCM and PchB) with the two substrates (chorismate and isochorismate) we show that the electrostatic field due to EcCM at atoms of chorismate favors the chorismate to prephenate transition and that, analogously, the electrostatic field due to PchB at atoms of isochorismate favors the isochorismate to salicylate transition. The largest differences between EcCM and PchB in electrostatic field strengths at atoms of the substrates are found to be due to residue side chains at distances between 0.6 and 0.8 nm from particular substrate atoms. Both enzymes tend to bring their non‐native substrate in the same conformation as their native substrate. EcCM and to a lower extent PchB fail in influencing the forces on and conformations of the substrate such as to favor the other chemical reaction (isochorismate pyruvate lyase activity for EcCM and chorismate mutase activity for PchB). These observations might explain the difficulty of engineering isochorismate pyruvate lyase activity in EcCM by solely mutating active site residues.     

Projected marine climate change: effects on copepod oxidative status and reproduction

Zooplankton are an important link between primary producers and fish. Therefore, it is crucial to address their responses when predicting effects of climate change on pelagic ecosystems. For realistic community‐level predictions, several biotic and abiotic climate‐related variables should be examined in combination. We studied the combined effects of ocean acidification and global warming predicted for year 2100 with toxic cyanobacteria on the calanoid copepod, Acartia bifilosa. Acidification together with higher temperature reduced copepod antioxidant capacity. Higher temperature also decreased egg viability, nauplii development, and oxidative status. Exposure to cyanobacteria and its toxin had a negative effect on egg production but, a positive effect on oxidative status and egg viability, giving no net effects on viable egg production. Additionally, nauplii development was enhanced by the presence of cyanobacteria, which partially alleviated the otherwise negative effects of increased temperature and decreased pH on the copepod recruitment. The interactive effects of temperature, acidification, and cyanobacteria on copepods highlight the importance of testing combined effects of climate‐related factors when predicting biological responses.     

Long-Term Prognostic Value of Restitution Slope in Patients with Ischemic and Dilated Cardiomyopathies

Background

An action potential duration (APD) restitution curve with a steep slope ≥1 has been associated with increased susceptibility for malignant ventricular arrhythmias. We aimed to evaluate the “restitution hypothesis” and tested ventricular APD restitution slope as well as effective refractory period (ERP)/APD ratio for long-term prognostic value in patients with ischemic (ICM) or dilated cardiomyopathy (DCM).

Methodology/Principal Findings

Monophasic action potentials were recorded in patients with ICM (n = 32) and DCM (n = 42) undergoing routine programmed ventricular stimulation (PVS). Left ventricular ejection fraction was 32±7% and 28±9%, respectively. APD and ERP were measured at baseline stimulation (S₁) and upon introduction of one to three extrastimuli (S₂–S₄). ERP/APD ratios and the APD restitution curve were calculated and the maximum restitution slope was determined. After a mean follow-up of 6.1±3.0 years, the combined end-point of mortality and and/or implantable cardioverter-defibrillator shock was not predicted by restitution slope or ERP/APD ratios. Comparing S₂ vs. S₃ vs. S₄ extrastimuli for restitution slope (1.5±0.6 vs. 1.4±0.4 vs. 1.3±0.5; p = NS), additional extrastimuli did not lead to a steepening restitution slope. ERP/APD ratio decreased with additional extrastimuli (0.98±0.09 [S₁] vs. 0.97±0.10 [S₂] vs. 0.93±0.11 [S₃]; p = 0.03 S₁ vs. S₃). Positive PVS was strongly predictive of outcome (p = 0.006).

Conclusions/Significance

Neither ventricular APD restitution slope nor ERP/APD ratios predict outcome in patients with ICM or DCM.     

How the Polls Can Be Both Spot On and Dead Wrong: Using Choice Blindness to Shift Political Attitudes and Voter Intentions

Political candidates often believe they must focus their campaign efforts on a small number of swing voters open for ideological change. Based on the wisdom of opinion polls, this might seem like a good idea. But do most voters really hold their political attitudes so firmly that they are unreceptive to persuasion? We tested this premise during the most recent general election in Sweden, in which a left- and a right-wing coalition were locked in a close race. We asked our participants to state their voter intention, and presented them with a political survey of wedge issues between the two coalitions. Using a sleight-of-hand we then altered their replies to place them in the opposite political camp, and invited them to reason about their attitudes on the manipulated issues. Finally, we summarized their survey score, and asked for their voter intention again. The results showed that no more than 22% of the manipulated replies were detected, and that a full 92% of the participants accepted and endorsed our altered political survey score. Furthermore, the final voter intention question indicated that as many as 48% (±9.2%) were willing to consider a left-right coalition shift. This can be contrasted with the established polls tracking the Swedish election, which registered maximally 10% voters open for a swing. Our results indicate that political attitudes and partisan divisions can be far more flexible than what is assumed by the polls, and that people can reason about the factual issues of the campaign with considerable openness to change.     

Inducible Arginase 1 Deficiency in Mice Leads to Hyperargininemia and Altered Amino Acid Metabolism

Arginase deficiency is a rare autosomal recessive disorder resulting from a loss of the liver arginase isoform, arginase 1 (ARG1), which is the final step in the urea cycle for detoxifying ammonia. ARG1 deficiency leads to hyperargininemia, characterized by progressive neurological impairment, persistent growth retardation and infrequent episodes of hyperammonemia. Using the Cre/loxP-directed conditional gene knockout system, we generated an inducible Arg1-deficient mouse model by crossing “floxed” Arg1 mice with CreER^T2 mice. The resulting mice (Arg-Cre) die about two weeks after tamoxifen administration regardless of the starting age of inducing the knockout. These treated mice were nearly devoid of Arg1 mRNA, protein and liver arginase activity, and exhibited symptoms of hyperammonemia. Plasma amino acid analysis revealed pronounced hyperargininemia and significant alterations in amino acid and guanidino compound metabolism, including increased citrulline and guanidinoacetic acid. Despite no alteration in ornithine levels, concentrations of other amino acids such as proline and the branched-chain amino acids were reduced. In summary, we have generated and characterized an inducible Arg1-deficient mouse model exhibiting several pathologic manifestations of hyperargininemia. This model should prove useful for exploring potential treatment options of ARG1 deficiency.     

High‐throughput sequencing and graph‐based cluster analysis facilitate microsatellite development from a highly complex genome

Despite recent advances in high‐throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club‐legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired‐end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild‐caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high‐throughput sequencing and graph‐based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.