The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikDFG, 53182490, EXC 314: Unifying Concepts in Catalysi

The study design of UDRIVE: the Naturalistic Driving Study across Europe for cars, trucks and scooters

Purpose: UDRIVE is the first large-scale European Naturalistic Driving Study on cars, trucks and powered two wheelers. The acronym stands for "European naturalistic Driving and Riding for Infrastructure & Vehicle safety and Environment". The purpose of the study is to gain a better understanding of what happens on the road in everyday traffic situations. Methods: The paper describes Naturalistic Driving Studies, a method which provides insight into the actual real-world behaviour of road users, unaffected by experimental conditions and related biases. Naturalistic driving can be defined as a study undertaken to provide insight into driver behaviour during everyday trips by recording details of the driver, the vehicle and the surroundings through unobtrusive data gathering equipment and without experimental control. Data collection will take place in six EU Member States. Results: Road User Behaviour will be studied with a focus on both safety and environment. The UDRIVE project follows the steps of the FESTA-V methodology, which was originally designed for Field Operational Tests. Conclusions: Defining research questions forms the basis of the study design and the specification of the recording equipment. Both will be described in this paper. Although the project has just started collecting data from drivers, we consider the process of designing the study as a major result which may help other initiatives to set up similar studies

Toxicity Testing in the 21st Century: Defining New Risk Assessment Approaches Based on Perturbation of Intracellular Toxicity Pathways

The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21st Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) “toxicity pathways” (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair

Perceived movement skill competence in stability: Validity and reliability of a pictorial scale in early adolescents

Perceived motor competence (PMC) is important to health as it mediates the association between actual motor competence (AMC) and physical activity. Many instruments assess the broader construct of physical self-perception but no scale has been developed to assess PMC in stability. The aim of this study was to develop and analyze the reliability and validity of a new pictorial PMC in stability skill assessment when completed by early adolescents. A Delphi method showed ≥70% of experts’ consensus in the seven proposed items. A sample of 904 students (11-14 years old) self-reported PMC in locomotion, object control, and stability using two pictorial scales: Perceived Movement Skill Competence (PMSC) and the newly developed Stability (PMSC_Stability). The Korperkoordinations test for Kinder (KTK) was also administered as a measure of AMC in skills that utilize stability. Two different subsamples were randomly assigned to criterion validity and reliability assessments. Spearman's correlation, confirmatory factor analysis, and a general linear model for sex and age were conducted. The internal consistency (α) and test-retest (intraclass correlation coefficient) reliability values were good (>0.70). A single-factor latent model represented stability and did not differ by sex. Stability perception was associated with AMC (rho = 0.51). Stability is important to the performance of many movement skills (eg, single-leg balance or moving on a narrow surface). Understanding which adolescents have poor stability perception could highlight stability as an area that needs targeting in order to increase physical activity and sport participation

Novel Extended Tetrathiafulvalenes Based on Acetylenic Spacers: Synthesis and Electronic Properties

A selection of mono- and diacetylenic dithiafulvalenes was synthesized and employed for the construction of extended tetrathiafulvalenes (TTFs) with hexa-2,4-diyne-1,6-diylidene or deca-2,4,6,8-tetrayne-1,10-diylidene spacers between the two 1,3-dithiole rings. By stepwise acetylenic scaffolding using (E)-1,2-diethynylethene (DEE) building blocks, an extended TTF containing a total of 18 C(sp) and C(sp2) atoms in the spacer was prepared. The versatility of the acetylenic dithiafulvene modules was also established by the efficient synthesis of a thiophenespaced TTF, employing a palladiumcatalyzed cross-coupling reaction. The developed synthetic protocols allow functionalization of the extended TTFs in three general ways: with 1) peripheral substituents on the fulvalene cores, 2) alkynyl moieties laterally appended to the spacer, and 3) cobalt clusters involving acetylenic moieties. Strong chromophoric properties of the extended TTFs were revealed by linear and nonlinear optical spectroscopies. Extensive electrochemical studies and calculations on these compounds are also reported, as well as X-ray crystallographic analyses

Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase

Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme