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Background: Childhood obesity is associated with various health outcomes. Restrictive measures to contain the spread of the Coronavirus Disease 2019 (COVID-19) pandemic, like lockdowns and school closures, affected children's daily structure, physical activity, dietary habits, and sleep quality, possibly exacerbating risk factors for childhood obesity and higher body mass index (BMI) in children. Poor socioeconomic conditions may have led to relatively higher risk for elevated BMI levels following pandemic measures. In this study, the impact of measures related to the COVID-19 pandemic on the BMI of third graders was investigated regarding children's socioeconomic background (SEB)., Methods: Data from 41,728 children (8.84 ± 0.56 years, 20,431 female) were collected in the context of a cohort study. Children were tested either before the pandemic (pre COVID : Sept2017-March2020, n = 26,314), or following the first (post LDI : Aug2020-Dec2020, n = 6657) or second lockdown in Germany (post LDII : Aug2021-Jan2022, n = 8757). SEB was based on the official school type classification of the state of Berlin. Outcome was BMI standard deviation scores (SDS)., Results: Significant effects of Time and SEB revealed elevated BMIs in post LDI (M = 0.23, p = 0.011) and post LDII (M = 0.22, p = 0.011) compared to pre COVID (M = 0.17) cohorts and higher BMIs for children with lower SEB (b = - 0.13, p < 0.001). A significant Time × SEB interaction indicated that the effect of SEB on children's BMI increased in response to lockdowns, especially in post LDII (b = - 0.05, p = 0.006). Results suggest that the COVID-19-related measures lead to increased BMI in children, and that children of lower SEB were at particular risk for higher BMIs following lockdowns., Conclusions: These findings highlight the dependency of children's BMI on societal circumstances. Over the course of two lockdowns in Germany, children have experienced BMI increments, particularly in low socioeconomic areas. Authorities are called into action to counteract increasing rates of childhood weight by promoting physical activity of children and establishing related post-pandemic offers., (© 2024. The Author(s).)

The electroenzymatic hydroxylation of 4-ethylbenzoic acid catalyzed by the recombinant unspecific peroxygenase from the fungus Agrocybe aegerita (rAaeUPO) was performed in a gas diffusion electrode (GDE)-based system. Enzyme stability and productivity are significantly affected by the way the co-substrate hydrogen peroxide (H 2 O 2 ) is supplied. In this study, two in-situ electrogeneration modes of H 2 O 2 were established and compared. Experiments under galvanostatic conditions (constant productivity of H 2 O 2 ) were conducted at current densities spanning from 0.8 mA cm -2 to 6.4 mA cm -2 . For comparison, experiments under H 2 O 2 -stat mode (constant H 2 O 2 concentration) were performed. Here, four H 2 O 2 concentrations between 0.06 mM and 0.28 mM were tested. A maximum H 2 O 2 productivity of 5.5 µM min -1 cm -2 and productivity of 10.5 g L -1 d -1 were achieved under the galvanostatic condition at 6.4 mA cm -2 . Meanwhile, the highest total turnover number (TTN) of 710,000 mol mol -1 and turnover frequency (TOF) of 87.5 s -1 were obtained under the H 2 O 2 -stat mode at concentration limits of 0.15 mM and 0.28 mM, respectively. The most favorable outcome in terms of maximum achievable TTN, TOF and productivity was found under the H 2 O 2 -stat mode at concentration limit of 0.2 mM. Here, a TTN of 655,000 mol mol -1 , a TOF of 80.3 s -1 and a productivity of 6.1 g L -1 d -1 were achieved. The electrochemical H 2 O 2 -stat mode not only offers a promising alternative reaction concept to the well-established galvanostatic mode but also enhances the process performance of unspecific peroxygenases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Light perception for orientation in zoospore-forming fungi is linked to homo- or heterodimeric rhodopsin-guanylyl cyclases (RGCs). Heterodimeric RGCs, first identified in the chytrid Rhizoclosmatium globosum , consist of an unusual near-infrared absorbing highly fluorescent sensitizer neorhodopsin (NeoR) that is paired with a visual light-absorbing rhodopsin responsible for enzyme activation. Here, we present a comprehensive analysis of the distribution of RGC genes in early-branching fungi using currently available genetic data. Among the characterized RGCs, we identified red-sensitive homodimeric RGC variants with maximal light activation close to 600 nm, which allow for red-light control of GTP to cGMP conversion in mammalian cells. Heterodimeric RGC complexes have evolved due to a single gene duplication within the branching of Chytridiales and show a spectral range for maximal light activation between 480 to 600 nm. In contrast, the spectral sensitivity of NeoRs is reaching into the near-infrared range with maximal absorption between 641 and 721 nm, setting the low energy spectral edge of rhodopsins so far. Based on natural NeoR variants and mutational studies, we reevaluated the role of the counterion-triad proposed to cause the extreme redshift. With the help of chimera constructs, we disclose that the cyclase domain is crucial for functioning as homo- or heterodimers, which enables the adaptation of the spectral sensitivity by modular exchange of the photosensor. The extreme spectral plasticity of retinal chromophores in native photoreceptors provides broad perspectives on the achievable spectral adaptation for rhodopsin-based molecular tools ranging from UVB into the near-infrared.

Rhomboid proteases hydrolyze substrate helices within the lipid bilayer to release soluble domains from the membrane. Here, we investigate the mechanism of activity regulation for this unique but wide-spread protein family. In the model rhomboid GlpG, a lateral gate formed by transmembrane helices TM2 and TM5 was previously proposed to allow access of the hydrophobic substrate to the shielded hydrophilic active site. In our study, we modified the gate region and either immobilized the gate by introducing a maleimide-maleimide (M2M) crosslink or weakened the TM2/TM5 interaction network through mutations. We used solid-state nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, and molecular docking to investigate the resulting effects on structure and dynamics on the atomic level. We find that variants with increased dynamics at TM5 also exhibit enhanced activity, whereas introduction of a crosslink close to the active site strongly reduces activity. Our study therefore establishes a strong link between the opening dynamics of the lateral gate in rhomboid proteases and their enzymatic activity.

Syngas fermentation processes with acetogens represent a promising process for the reduction of CO 2 emissions alongside bulk chemical production. However, to fully realize this potential the thermodynamic limits of acetogens need to be considered when designing a fermentation process. An adjustable supply of H 2 as electron donor plays a key role in autotrophic product formation. In this study an anaerobic laboratory scale continuously stirred tank reactor was equipped with an All-in-One electrode allowing for in-situ H 2 generation via electrolysis. Furthermore, this system was coupled to online lactate measurements to control the co-culture of a recombinant lactate-producing Acetobacterium woodii strain and a lactate-consuming Clostridium drakei strain to produce caproate. When C. drakei was grown in batch cultivations with lactate as substrate, 1.6 g·L -1 caproate were produced. Furthermore, lactate production of the A. woodii mutant strain could manually be stopped and reinitiated by controlling the electrolysis. Applying this automated process control, lactate production of the A. woodii mutant strain could be halted to achieve a steady lactate concentration. In a co-culture experiment with the A. woodii mutant strain and the C. drakei strain, the automated process control was able to dynamically react to changing lactate concentrations and adjust H 2 formation respectively. This study confirms the potential of C. drakei as medium chain fatty acid producer in a lactate-mediated, autotrophic co-cultivation with an engineered A. woodii strain. Moreover, the monitoring and control strategy presented in this study reinforces the case for autotrophically produced lactate as a transfer metabolite in defined co-cultivations for value-added chemical production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Herzog, Mook, Utesch, Bengelsdorf and Zeng.)

Writing presents considerable challenges to students' motivation. Yet there is a dearth of studies assessing the role of affect and motivation in writing performance for students with migration backgrounds (MB), who often underachieve in writing. Our study addressed this research gap by investigating the interplay between writing self-efficacy, writing anxiety, and text quality in 208 secondary students with and without MB using Response Surface Analyses. The data showed comparable levels of self-efficacy and, notably, lower writing anxiety levels among students with MB despite lower writing achievements. In the full sample, we observed positive correlations between self-efficacy and text quality and negative correlations between writing anxiety and text quality. When modeling efficacy and anxiety measures and their interplay to predict text quality, self-efficacy measures continued to account for statistically detectable unique variance in text quality, whereas writing anxiety did not. However, students with MB demonstrated differing interplay patterns, with less efficacious students with MB showing positive relations between writing anxiety and text quality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Busse, Graham, Müller and Utesch.)

Membrane models assembled on electrodes are widely used tools to study potential-dependent molecular processes at or in membranes. However, the relationship between the electrode potential and the potential across the membrane is not known. Here we studied lipid bilayers immobilized on mixed self-assembled monolayers (SAM) on Au electrodes. The mixed SAM was composed of thiol derivatives of different chain lengths such that between the islands of the short one, mercaptobenzonitrile (MBN), and the tethered lipid bilayer an aqueous compartment was formed. The nitrile function of MBN, which served as a reporter group for the vibrational Stark effect (VSE), was probed by surface-enhanced infrared absorption spectroscopy to determine the local electric field as a function of the electrode potential for pure MBN, mixed SAM, and the bilayer system. In parallel, we calculated electric fields at the VSE probe by molecular dynamics (MD) simulations for different charge densities on the metal, thereby mimicking electrode potential changes. The agreement with the experiments was very good for the calculations of the pure MBN SAM and only slightly worse for the mixed SAM. The comparison with the experiments also guided the design of the bilayer system in the MD setups, which were selected to calculate the electrode potential dependence of the transmembrane potential, a quantity that is not directly accessible by the experiments. The results agree very well with estimates in previous studies and thus demonstrate that the present combined experimental-theoretical approach is a promising tool for describing potential-dependent processes at biomimetic interfaces.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity., (© 2022. The Author(s).)

Background: Electro-fermentation (EF) is an emerging tool for bioprocess intensification. Benefits are especially expected for bioprocesses in which the cells are enabled to exchange electrons with electrode surfaces directly. It has also been demonstrated that the use of electrical energy in BES can increase bioprocess performance by indirect secondary effects. In this case, the electricity is used to alter process parameters and indirectly activate desired pathways. In many bioprocesses, oxidation-reduction potential (ORP) is a crucial process parameter. While C. pasteurianum fermentation of glycerol has been shown to be significantly influenced electrochemically, the underlying mechanisms are not clear. To this end, we developed a system for the electrochemical control of ORP in continuous culture to quantitatively study the effects of ORP alteration on C. pasteurianum by metabolic flux analysis (MFA), targeted metabolomics, sensitivity and regulation analysis., Results: In the ORP range of -462 mV to -250 mV, the developed algorithm enabled a stable anodic electrochemical control of ORP at desired set-points and a fixed dilution rate of 0.1 h -1 . An overall increase of 57% in the molar yield for 1,3-propanediol was observed by an ORP increase from -462 to -250 mV. MFA suggests that C. pasteurianum possesses and uses cellular energy generation mechanisms in addition to substrate-level phosphorylation. The sensitivity analysis showed that ORP exerted its strongest impact on the reaction of pyruvate-ferredoxin-oxidoreductase. The regulation analysis revealed that this influence is mainly of a direct nature. Hence, the observed metabolic shifts are primarily caused by direct inhibition of the enzyme upon electrochemical production of oxygen. A similar effect was observed for the enzyme pyruvate-formate-lyase at elevated ORP levels., Conclusions: The results show that electrochemical ORP alteration is a suitable tool to steer the metabolism of C. pasteurianum and increase product yield for 1,3-propanediol in continuous culture. The approach might also be useful for application with further anaerobic or anoxic bioprocesses. However, to maximize the technique's efficiency, it is essential to understand the chemistry behind the ORP change and how the microbial system responds to it by transmitted or direct effects., (© 2022. The Author(s).)

Purpose: Movement competence is a key outcome for primary physical education (PE) curricula. As movement development in children emerges through physical activity (PA), it is important to determine the extent of PA promotion within movement competence focused teaching pedagogies. Therefore, this study aimed to assess children's moderate-to-vigorous PA (MVPA) and related teaching practices in primary PE within Linear pedagogy and Nonlinear pedagogy and to compare this to current practice within PE delivery in primary schools., Methods: Participants (n = 162, 53% females, 5-6y) were recruited from 9 primary schools within the SAMPLE-PE cluster randomised controlled trial. Schools were randomly-allocated to one of three conditions: Linear pedagogy, Nonlinear pedagogy, or control. Nonlinear and Linear pedagogy intervention schools received a PE curriculum delivered by trained deliverers over 15 weeks, while control schools followed usual practice. Children's MVPA was measured during 3 PE lessons (44 PE lessons in total) using an ActiGraph GT9X accelerometer worn on their non-dominant wrist. Differences between conditions for children's MVPA were analysed using multilevel model analysis. Negative binomial models were used to analyse teaching practices data., Results: No differences were found between Linear pedagogy, Nonlinear pedagogy and the control group for children's MVPA levels during PE. Linear and Nonlinear interventions generally included higher percentages of MVPA promoting teaching practices (e.g., Motor Content) and lower MVPA reducing teaching practices (e.g., Management), compared to the control group. Teaching practices observed in Linear and Nonlinear interventions were in line with the respective pedagogical principles., Conclusions: Linear and Nonlinear pedagogical approaches in PE do not negatively impact MVPA compared to usual practice. Nevertheless, practitioners may need to refine these pedagogical approaches to improve MVPA alongside movement competence., Competing Interests: The authors have declared that no competing interests exist.

A novel approach of phenotype analysis of fermentation-based bioprocesses based on unsupervised learning (clustering) is presented. As a prior identification of phenotypes and conditional interrelations is desired to control fermentation performance, an automated learning method to output reference phenotypes (defined as vector of biomass-specific rates) was developed and the necessary computing process and parameters were assessed. For its demonstration, time series data of 90 Clostridium pasteurianum cultivations were used which feature a broad spectrum of solventogenic and acidogenic phenotypes, while 14 clusters of phenotypic manifestations were identified. The analysis of reference phenotypes showed distinct differences, where potential conditionalities were exemplary isolated. Further, cluster-based balancing of carbon and ATP or the use of reference phenotypes as indicator for bioprocess monitoring were demonstrated to highlight the perks of this approach. Overall, such analysis depends strongly on the quality of the data and experimental validations will be required before conclusions. However, the automated, streamlined and abstracted approach diminishes the need of individual evaluation of all noisy dataset and showed promising results, which could be transferred to strains with comparably wide-ranging phenotypic manifestations or as indicators for repeated bioprocesses with clearly defined target., Competing Interests: The authors have declared no conflict of interests., (© 2021 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

Intramembrane proteolysis plays a fundamental role in many biological and pathological processes. Intramembrane proteases thus represent promising pharmacological targets, but few selective inhibitors have been identified. This is in contrast to their soluble counterparts, which are inhibited by many common drugs, and is in part explained by the inherent difficulty to characterize the binding of drug-like molecules to membrane proteins at atomic resolution. Here, we investigated the binding of two different inhibitors to the bacterial rhomboid protease GlpG, an intramembrane protease characterized by a Ser-His catalytic dyad, using solid-state NMR spectroscopy. H/D exchange of deuterated GlpG can reveal the binding position while chemical shift perturbations additionally indicate the allosteric effects of ligand binding. Finally, we determined the exact binding mode of a rhomboid protease-inhibitor using a combination of solid-state NMR and molecular dynamics simulations. We believe this approach can be widely adopted to study the structure and binding of other poorly characterized membrane protein-ligand complexes in a native-like environment and under physiological conditions., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are densely phosphorylated eukaryotic messengers, which are involved in numerous cellular processes. To elucidate their signaling functions at the molecular level, non-hydrolyzable bisphosphonate analogs of inositol pyrophosphates, PCP-InsPs, have been instrumental. Here, an efficient synthetic strategy to obtain these analogs in unprecedented quantities is described - relying on the use of combined phosphate ester-phosphoramidite reagents. The PCP-analogs, alongside their natural counterparts, were applied to investigate their regulatory effect on insulin-degrading enzyme (IDE), using a range of biochemical, biophysical and computational methods. A unique interplay between IDE, its substrates and the PP-InsPs was uncovered, in which the PP-InsPs differentially modulated the activity of the enzyme towards short peptide substrates. Aided by molecular docking and molecular dynamics simulations, a flexible binding mode for the InsPs/PP-InsPs was identified at the anion binding site of IDE. Targeting IDE for therapeutic purposes should thus take regulation by endogenous PP-InsP metabolites into account., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

This study aimed to examine the associations between play behaviors during preschool recess and foundational movement skills (FMS) in typically developing preschool children. One hundred and thirty-three children (55% male; mean age 4.7 ± 0.5 years) from twelve preschools were video-assessed for six locomotor and six object-control FMS using the Champs Motor Skill Protocol. A modified System for Observing Children's Activity and Relationships during Play assessed play behaviors during preschool recess. Associations between the composition of recess play behaviors with FMS were analyzed using compositional data analysis and linear regression. Results: Relative to time spent in other types of play behaviors, time spent in play without equipment was positively associated with total and locomotor skills, while time spent in locomotion activities was negatively associated with total and locomotor skills. No associations were found between activity level and group size play behavior compositions and FMS. The findings suggest that activity type play behaviors during recess are associated with FMS. While active games without equipment appear beneficial, preschool children may need a richer playground environment, including varied fixed and portable equipment, to augment the play-based development of FMS.

Background: School-based interventions are a key opportunity to improve children's physical activity (PA); however, there is lack of evidence about how pedagogical approaches to motor learning in physical education (PE) might affect PA in children. Therefore, this study aimed to assess how different pedagogical approaches in PE might affect children's PA., Methods: Participants ( n = 360, 5-6 years) from 12 primary schools within the SAMPLE-PE randomized controlled trial were randomly allocated to either Linear Pedagogy (LP: n = 3) or Nonlinear Pedagogy (NP: n = 3) interventions, where schools received a 15-week PE intervention delivered by trained coaches, or to a control group ( n = 6), where schools followed usual practice. ActiGraph GT9X accelerometers were used to assess PA metrics (moderate-to-vigorous PA, mean raw acceleration and lowest acceleration over the most active hour and half hour) over whole and segmented weeks at baseline, immediately post-intervention and 6 months follow-up. Intention to treat analysis employing multilevel modelling was used to assess intervention effects., Results: LP and NP interventions did not significantly affect children's PA levels compared to the control group., Conclusion: PE interventions based on LP and NP alone might not be effective in improving habitual PA in children.

Lack of physical activity is a global public health problem causing not only morbidity and premature mortality, but it is also a major economic burden worldwide. One of the cornerstones of a physically active lifestyle is Motor Competence (MC). MC is a complex biocultural attribute and therefore, its study requires a multi-sectoral, multi-, inter- and transdisciplinary approach. MC is a growing area of research, especially in children and adolescents due to its positive association with a plethora of health and developmental outcomes. Many questions, however, remain to be answered in this field of research, with regard to: (i) Health and Developmental-related Associations of MC; (ii) Assessment of MC; (iii) Prevalence and Trends of MC; (iv) Correlates and Determinants of MC; (v) MC Interventions, and (vi) Translating MC Research into Practice and Policy. This paper presents a narrative review of the literature, summarizing current knowledge, identifying key research gaps and presenting questions for future investigation on MC in children and adolescents. This is a collaborative effort from the International Motor Competence Network (IMCNetwork) a network of academics and researchers aiming to promote international collaborative research and knowledge translation in the expansive field of MC. The knowledge and deliverables generated by addressing and answering the aforementioned research questions on MC presented in this review have the potential to shape the ways in which researchers and practitioners promote MC and physical activity in children and adolescents across the world., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

In this contribution, we studied the effect of electro-fermentation on the butanol production of Clostridium pasteurianum strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3-propanediol (PDO). The dhaB-negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway. Both strains showed a 3-5 fold increase of the intracellular NADH/NAD ratio when exposed to cathodic current in a bioelectrochemical system (BES). This drove an activation of the butanol pathway and resulted in a higher molar butanol to PDO ratio for the R525 strain. Nonetheless, macroscopic electron balances suggest that no significant amount of electrons derived from the BES was harvested by the cells. Overall, this work points out that electro-fermentation can be used to trigger metabolic pathways and improve product formation, even when the used microbe cannot be considered electroactive. Accordingly, further studies are required to unveil the underlying (regulatory) mechanisms., Competing Interests: The authors have declared no conflicts of interest., (© 2020 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

Introduction: Monitoring of physical fitness in youth is important because physical fitness is a summative indicator of health. From a developmental and preventive perspective, physical fitness levels are relatively stable from childhood to early adulthood. Thus, it is important to monitor physical fitness on a population based level being able to intervene at early stages (1). In order to reliably assess and evaluate the physical fitness of youth, a reliable system of standard values based on representative data is required. The aim of this analysis is to report sex- and age-specific physical fitness percentile curves from childhood to early adulthood in a nationwide sample in Germany. Methods: We use data from the nationwide representative Motorik Modul (MoMo) Study in Germany (data collection wave 1: 2009-2012; age: 4-23 years; n = 3,742; 50.1% female). Physical fitness was assessed by means of the MoMo test profile covering four dimensions of physical fitness (strength, endurance, coordination, and flexibility) and including eight physical fitness items. Percentile curves were fitted using the LMS transformation method of Cole and Green. Results: Standardized age- and sex-specific physical fitness percentiles were calculated for eight items: ergometric endurance testing, standing long jump, push-ups, sit-ups, jumping side-ways, balancing backwards, static stand, and stand and reach test. The physical fitness curves differ according to gender and the fitness dimension. Physical fitness improvements with age are linear (e.g., max. strength) or curvilinear (e.g., coordination) and have their stagnation points at different times over the course of adolescence. Discussion: Our results provide for the first time sex- and age-specific physical fitness percentile curves for Germany from 4 to 17 years. Differences in curve-shapes indicating a timed and capacity-specific physical fitness development. Nationwide German physical fitness percentiles can be useful in comparing different populations (e.g., cross-country), reporting secular trends, comparing special groups, and to evaluate physical fitness interventions., (Copyright © 2020 Niessner, Utesch, Oriwol, Hanssen-Doose, Schmidt, Woll, Bös and Worth.)

Background: There is a need for interdisciplinary research to better understand how pedagogical approaches in primary physical education (PE) can support the linked development of physical, cognitive and affective aspects of physical literacy and physical activity behaviors in young children living in deprived areas. The Skill Acquisition Methods fostering Physical Literacy in Early-Physical Education (SAMPLE-PE) study aims to examine the efficacy of two different pedagogies for PE, underpinned by theories of motor learning, to foster physical literacy., Methods: SAMPLE-PE will be evaluated through a cluster-randomized controlled trial targeting 5-6 year old children from schools located in areas of high deprivation in Merseyside, North-West England. Schools will be randomly allocated to one of three conditions: Linear Pedagogy , Non-linear Pedagogy , or Control. Non-linear and Linear Pedagogy intervention primary schools will receive a PE curriculum delivered by trained coaches over 15 weeks, while control schools will follow their usual practice. Data will be collected at baseline (T0), immediately post-intervention (T1), and 6 months after the intervention has finished (T2). Children's movement competence is the primary outcome in this trial. Secondary outcomes include physical activity, perceived competence, motivation, executive functions, and self-regulation. An extensive process evaluation will also examine implementation factors such as intervention context, reach, dose, fidelity and acceptability., Discussion: The SAMPLE-PE project will enable better understanding surrounding how to operationalise physical literacy through enrichment of PE practices in early PE. The study will provide robust scientific evidence regarding the efficacy of underpinning PE pedagogy with theories of motor learning to promote the development of physical literacy., Trial Registration: Retrospectively registered on 5th September 2018 at ClinicalTrials.gov, a resource provided by the U.S. National Library of Medicine (Identifier: NCT03551366)., (Copyright © 2020 Rudd, Crotti, Fitton-Davies, O’Callaghan, Bardid, Utesch, Roberts, Boddy, Cronin, Knowles, Foulkes, Watson, Pesce, Button, Lubans, Buszard, Walsh and Foweather.)

The Self-Determination Theory has been applied to explain behaviour in numerous contexts and cultures. In the exercise context, causality orientations (autonomy, control, impersonal) are important to describe individual differences in initiation and maintenance of health behaviour. The assessment of exercise causality orientations can be a key element to improve predictions of motivated health and exercise behaviour. Nevertheless, a scale to measure exercise causality orientations has not been established in German yet. Thus, it was the aim of the present work to translate the Exercise Causality Orientations Scale to German and to test it throughout three studies. The German G-ECOS questionnaire was cross-validated via confirmatory factor analyses in two separate samples. Both Study 1 (n = 306, 72.60% female, age M = 26.00, SD = 5.66; CFI = .96) and Study 2 (n = 320, 70.94% female, age M = 29.00, SD = 3.54; CFI = .95) indicated a good model fits. In a further Study 3 (n = 548, 62.50% female, age M = 30.17, SD = 11.91), the relations between exercise causality orientations and other SDT related constructs were examined. The correlations indicated positive associations between autonomy causality orientation and intrinsic regulation, intrinsic exercise participation goals, and exercise basic needs satisfaction. Overall, the assessment of exercise causality orientations can be useful in analysing and potentially predicting motivated exercise behaviour., Competing Interests: The authors have declared that no competing interests exist.

Background: Mental practice refers to the imaginary representation of a motor action. Mental practice interventions are frequently used among stroke survivors to improve motor function. Individual characteristics that may determine whether a person is able to mentally perform a specific movement have been mainly spared in research. Aims: The aim of the present study is to examine whether gender and age are related to mental practice ability. Methods: The study has a cross-sectional design. Data collection was done via self-report questionnaires on mental practice ability, sociodemographic information, and perceived stroke impact. Data analysis was conducted in R using descriptive statistics and regression analysis. N = 44 stroke survivors ( M = 65.8 years, SD = 11.4, range 48-88), n = 19 of which were female were recruited in two German neurologic rehabilitation facilities. Results: Age ( β = -0.13, p = 0.057) and gender ( β = 0.17, p = 0.260) were not associated with mental practice ability, when controlling for time since stroke and perceived stroke impact (Stroke Impact Scale). Perceived stroke impact was significantly related to mental practice ability ( β = 0.44, p = 0.004). Those who reported less stroke impact showed better mental practice ability. Conclusion: Mental practice ability may be preserved in stroke patients, irrespective of age and gender. We report cross-sectional data on mental practice ability in this study, thus the direction of the relationship between mental practice ability and perceived stroke impact is of interest. Future studies should aim at using a longitudinal design and bigger sample sizes.

Clostridium pasteurianum produces industrially valuable chemicals such as n -butanol and 1,3-propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit ( dhaB ) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3-propanediol-deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl-CoA to butyryl-CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl-CoA to butyryl-CoA and butanol, indicating a new, 1,3-propanediol-independent pattern of glycerol fermentation in Clostridium pasteurianum ., Competing Interests: The authors have declared no conflict of interest., (© 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP from Aspergillus giganteus , is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity. IMPORTANCE Fungal pathogens pose a serious danger to human welfare since they kill more people per year than malaria or tuberculosis and are responsible for crop losses worldwide. The treatment of fungal infections is becoming more complicated as fungi develop resistances against commonly used fungicides. Therefore, discovery and development of novel antifungal agents are of utmost importance., (Copyright © 2018 Utesch et al.)

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ E ads due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ E int , representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ E ads and Δ E int , determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ E ads ) and attached to membranes (Δ E ads + Δ E int ).

An autoclavable All-in-One electrolysis electrode in a rod shape assembly is developed as a new tool for bioelectrochemical systems and electricity-aided bioprocesses. It can replace the classic two-chamber bioelectrochemical system for electrolysis reactions, be inserted into conventional bioreactors and is easily adaptable as electrocatalytic surface or generator of super-fine bubbles (H 2 and O 2 ) for bioconversion processes. Whereas the bioreactor itself functions as the working electrode chamber, a well-integrated inner counter electrode chamber enables water electrolysis without the normally encountered undesired ion-transfer effect. The efficiencies of the electrode are characterized and its advantages and usefulness compared to the classic H-Cell bioelectrochemical system (BES) are demonstrated with glycerol fermentations by Clostridium pasteurianum DSM 525., (© 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

[NiFe] hydrogenases catalyze the reversible splitting of H 2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O 2 , a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha , is able to overcome aerobic inactivation by catalytic reduction of O 2 to water. This O 2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O 2 attack. Here, the O 2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O 2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O 2 concentrations used for MBH crystal derivatization. The examination of the O 2 -derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O 2 tolerance of the enzyme., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Two variants of the two-component Lantibiotic Lichenicidin, produced by the strains B. Licheniformis VK21 and I89 (Lchα/ Lchβ and Bliα/ Bliβ peptides, respectively) have been investigated by means of 2 μs-long all-atom molecular dynamics simulations combined with Markov State Models. This rigorous statistical analysis enabled to evaluate the dynamic and kinetic properties of the aforementioned systems which are not accessible via experimental techniques. The structural flexibility characteristic of these small peptides is understood by a delicate equilibrium between random coil, α-helices and β-sheet structures. The undergoing secondary structure transitions from an α-helix to a β-sheet observed for Lchα and Lchβ peptides, were not present in the Bliα component and provide new insights to understand their mechanism of action.

Hexameric tyrosine-coordinated heme protein HTHP from Silicibacter pomeroyi has been shown to exhibit peroxidase- and catalase-like activity. In the present study, detailed spectroscopic and electrochemical investigations were performed to analyze the redox properties and active site structure of HTHP. Potentiometric titration of HTHP in solution revealed a single redox transition at -0.54 V (vs Ag/AgCl), indicating six structurally identical tyrosine coordinates hemes. Cyclic voltammetry (CV) of immobilized HTHP afforded a distinctly more positive redox potential (-0.17 V) but failed to detect a transition at -0.54 V. Conversely, surface enhanced RR (SERR) spectroscopy provided evidence for both high- and low-potential transitions and for a partial loss of heme in the reduced state. The high-potential CV-active redox transition is attributed to the hemes of the barrel-shaped HTHP in a wheel-like orientation on the surface. Supported by coarse-grained simulations and SERR spectroscopy, the majority of HTHP is concluded to adopt a reverse-disc orientation, accounting for the low-potential transition. In view of the striking similarity of HTHP to the heme carriers HasA or HmbR regarding redox potential, Fe-Tyr ligation, and heme release, we propose heme transport as an alternative or additional function.

Background: Clostridium pasteurianum as an emerging new microbial cell factory can produce both n-butanol (BuOH) and 1,3-propanediol (1,3-PDO), and the pattern of product formation changes significantly with the composition of the culture medium. Among others iron content in the medium was shown to strongly affect the products selectivity. However, the mechanism behind this metabolic regulation is still unclear. For a better understanding of such metabolic regulation and for process optimization, we carried out fermentation experiments under either iron excess or iron limitation conditions, and performed metabolic, stoichiometric and proteomic analyses., Results: 1,3-PDO is most effectively produced under iron limited condition (Fe-), whereas 1,3-PDO and BuOH were both produced under iron rich condition (Fe+). With increased iron availability the BuOH/1,3-PDO ratio increased significantly from 0.27 mol/mol (at Fe-) to 1.4 mol/mol (at Fe+). Additionally, hydrogen production was enhanced significantly under Fe+ condition. Proteomic analysis revealed differentiated expression of many proteins including several ones of the central carbon metabolic pathway. Among others, pyruvate: ferredoxin oxidoreductase, hydrogenases, and several electron transfer flavoproteins was found to be strongly up-regulated under Fe+ condition, pointing to their strong involvement in the regeneration of the oxidized form of ferredoxin, and consequently their influences on the product selectivity in C. pasteurianum. Of particular significance is the finding that H 2 formation in C. pasteurianum is coupled to the ferredoxin-dependent butyryl-CoA dehydrogenase catalyzed reaction, which significantly affects the redox balance and thus the product selectivity., Conclusions: The metabolic, stoichiometric and proteomic results clearly show the key roles of hydrogenases and ferredoxins dependent reactions in determining the internal redox balance and hence product selectivity. Not only the NADH pool but also the regulation of the ferredoxin pool could explain such product variation under different iron conditions.

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.

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.

In the voltage-sensitive phosphatase Ci-VSP, conformational changes in the transmembrane voltage sensor domain (VSD) are transduced to the intracellular catalytic domain (CD) leading to its dephosphorylation activity against membrane-embedded phosphoinositides. The linker between both domains is proposed to be crucial for the VSD-CD coupling. With a combined approach of electrophysiological measurements on Xenopus oocytes and molecular dynamics simulations of a Ci-VSP model embedded in a lipid bilayer, we analyzed how conformational changes in the linker mediate the interaction between the CD and the activated VSD. In this way, we identified specific residues in the linker that interact with well-defined amino acids in one of the three loops forming the active site of the protein, named TI loop. With our results, we shed light into the early steps of the coupling process between the VSD and the CD, which are based on fine-tuned electrostatic and hydrophobic interactions between the linker, the membrane and the CD.

Post-translational nitration of tyrosine is considered to be an important step in controlling the multiple functions of cytochrome c (Cyt-c). However, the underlying structural basis and mechanism are not yet understood. In this work, human Cyt-c variants in which all but one tyrosine has been substituted by phenylalanine have been studied by resonance Raman and electrochemical methods to probe the consequences of tyrosine nitration on the heme pocket structure and the redox potential. The mutagenic modifications of the protein cause only subtle conformational changes of the protein and small negative shifts of the redox potentials which can be rationalized in terms of long-range electrostatic effects on the heme. The data indicate that the contributions of the individual tyrosines for maintaining the relatively high redox potential of Cyt-c are additive. Nitration of individual tyrosines leads to a destabilization of the axial Fe-Met80 bond which causes the substitution of the native Met ligand by a water molecule or a lysine residue for a fraction of the proteins. Electrostatic immobilization of the protein variants on electrodes coated by self-assembled monolayers (SAMs) of mercaptounadecanoic acid destabilizes the heme pocket structure of both the nitrated and non-nitrated variants. Here, the involvement of surface lysines in binding to the SAM surface prevents the replacement of the Met80 ligand by a lysine but instead a His-His coordinated species is formed. The results indicate that structural perturbations of the heme pocket of Cyt-c due to tyrosine nitration and to local electric fields are independent of each other and occur via different molecular mechanisms. The present results are consistent with the view that either tyrosine nitration or electrostatic binding to the inner mitochondrial membrane, or both events together, are responsible for the switch from the redox to the peroxidase function.

The voltage-sensitive phosphatase Ci-VSP consists of an intracellular phosphatase domain (PD) coupled to a transmembrane voltage-sensor domain (VSD). Depolarization triggers the selective dephosphorylation of phosphoinositides. However, the molecular mechanisms of coupling are still elusive. To clarify the role of the VSD-PD linker as a putative partner for electrostatic interactions with the membrane, we carried out a cysteine-scanning mutagenesis of the whole motif M240-K257. Upon coexpression with PI(4,5)P(2)-sensitive KCNQ2/KCNQ3 channels in Xenopus oocytes, we identified four positions (A242C, R245C, K252C, and Y255C) with a completely abrogated PD activity. Because the mutation effect occurred periodically, we hypothesize that α-helical elements exist within the linker, with a gap near position S249. The combination of these results with the analysis of transient sensing currents of the VSD revealed distinct roles for the N-terminal (M240-S249) and C-terminal (Q250-K257) linker motifs in the VSD-PD coupling. According to our functional results, the computational structure prediction of the Q239-D258 fragment confirmed α-helical structures within the linker, with a short β-turn around S249 in the activated conformation. Remarkably, the position K252 may be a candidate for interacting with the PD rather than for binding to the membrane. This provides the first insight (to our knowledge) into the direct intervention of the linker in the VSD-PD coupling process., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode., (© 2011 American Chemical Society)

The catalytic cycle of the anaerobic [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) both in solution and immobilized on an Au electrode was studied by IR spectroscopic and electrochemical methods. IR spectroelectrochemistry in solution at different pH values allows the identification of the various redox-states of the active site and the determination of the midpoint potentials, as well as their acid-base equilibria. The spectroscopic characterization was based on the unique marker bands of the CN and CO stretching modes of the Ni-Fe center and served as reference for the surface-enhanced IR absorption (SEIRA) study of the immobilized enzyme. Using structural models of hydrogenases from DvMF and Desulfovibrio gigas , dipole moment calculations were carried out to guide the immobilization strategy. In view of the high dipole moment of about 1100 D pointing through the negatively charged area surrounding the distal [FeS] cluster, the Au electrode was coated by a self-assembled monolayer of amino-terminated mercaptanes which, due to the positively charged head groups, permit a durable electrostatic binding of the protein. SEIRA spectroscopy revealed a structurally and functionally intact active site as demonstrated by the reversible activation and inactivation under hydrogen and argon, respectively. Cyclic voltammetry on the immobilized enzyme demonstrate a reversible anaerobic inactivation upon changing the applied potential. The "switch" potential (E(switch)) associated with the reductive reactivation was determined to be -33 mV (vs normal hydrogen electrode). However, the catalytic current decreased on the time scale of hours during continuous cycling. SEIRA experiments demonstrate that the loss of catalytic activity is not due to protein desorption but is rather related to a slow degradation of the active site, possibly initiated by the attack of reactive species electrochemically generated from residual traces of oxygen in solution.

Iso-1 yeast cytochrome c (YCC) was adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) consisting either of 11-mercaptoundecanoic acid (MUA) or of 1:1 mixtures of MUA and either 11-mercaptoundecanol (MU) or 7-mercaptoheptanol (MH). The redox potentials and the apparent rate constants for the interfacial redox process as well as for the protein reorientation were determined by stationary surface-enhanced resonance Raman (SERR) and time-resolved SERR spectroscopy, respectively. For YCC immobilized on MUA and MUA/MU at pH 7.0 and 6.0, the negative shifts of the redox potentials with respect to that for the protein in solution can be rationalized in terms of the potential of the zero-charge determined by impedance measurements. The apparent electron transfer rate constants of YCC on MUA/MU and MU/MH at pH 6.0 were determined to be 8 and 18 s(-1), respectively. A decrease of the relaxations constants by a factor of ca. 2 was found for pH 7.0, and a comparable low value was determined for a pure MUA even at pH 6.0. In each system, the rate constant for protein reorientation was found to be the same as that for the electron transfer, implying that protein reorientation is the rate limiting step for the interfacial redox process. This gating step is distinctly slower than that for horse heart cytochrome c (HHCC) observed previously under similar conditions (Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062-4068). The different rate constants of protein reorientation for both proteins and the variations of the rate constants for the different SAMs and pH are attributed to the electric field dependence of the free energy of activation which is assumed to be proportional to the product of the electric field strength and the molecular dipole moment of the protein. The latter quantity is determined by molecular dynamics simulations and electrostatic calculations to be more than 2 times larger for YCC than for HHCC. Moreover, the dipole moment vector and the heme plane constitute an angle of ca. 10 and 45 degrees in YCC and HHCC, respectively. The different magnitudes and directions of the dipole moments as well as the different electric field strengths at the various SAM/protein interfaces allow for a qualitative description of the protein-, SAM-, and electrode-specific kinetics of the interfacial redox processes studied in this and previous works.