Search

Background: Time constraints comprise one limiting factor for implementing school-based physical activity programs. The aim of this pilot cluster randomized controlled study was to explore the effects of a cycle ergometer intervention during regular lessons on physical fitness, body composition, and health-related blood parameters. Methods: Participants attended one of 2 classes selected from one school, which were randomly assigned to an intervention group (n = 23, 11.2 [0.5] y) consisting of cycling on classroom-based ergometers during 3 lessons per week at a self-selected intensity and a control group (n = 21, 11.3 [0.5] y) not receiving any treatment. Prior to and after the 5-month intervention period, physical fitness (with ventilatory threshold as primary outcome), body composition, and parameters of glucose and lipid metabolism were assessed. Results: A significant time × group interaction was revealed for ventilatory threshold (P = .035), respiratory compensation point (P = .038), gross efficiency (P P = .024), triglycerides (P = .041), and blood glucose levels (P = .041) with benefits for the intervention group. Peak oxygen uptake and body composition were not affected. Conclusions: Children’s aerobic capacity benefited from the low-intensity school-based cycling intervention, while body composition and most blood parameters were not affected. The intervention using cycle ergometers is a feasible and time-saving strategy to elevate submaximal physical fitness.

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein–carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM–substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose–CBM bond rupture forces exceeding 15 pN.

The cooperative effect of multiple affinity binding interactions creating a stable bond, known as avidity, is particularly important in assessing the potency of potential drugs such as monoclonal antibodies, CAR T, or NK cells to treat cancer. However, predicting avidity based onin vitrosingle affinity interactions has limitations and often fails to describe the avidity effects observedin vivo. Acoustic force-based assays have recently emerged as a reliable method for direct avidity measurements, expressed as adhesion forces, which positively correlate with drug efficacy. However, to better understand avidity, in particular for cell-cell interactions and correlate it with affinity, a cell model system with controlled avidity-related properties is needed. This study presents a method for producing a cell model system using “effector beads” that can be used in acoustic force spectroscopy-based avidity assays or any other bead-based avidity assay. The protein of interest is biotinylatedin vivoinE.coli, purified and subsequently mixed with streptavidin coated beads to create effector beads. The results demonstrate the dependency of rupture force on the receptor surface density and force loading rate, thus providing valuable information for designing future effector bead assays as well as cell avidity measurements for screening and characterization purposes.

The production of biofuels from lignocellulosic biomass using carbohydrate-active enzymes like cellulases is key to sustainable energy production. Understanding the adsorption mechanism of cellulases and associated binding domain proteins down to the molecular level details will help in the rational design of improved cellulases. In nature, carbohydrate-binding modules (CBMs) from families 17 and 28 often appear in tandem appended to the C-terminus of several endocellulases. Both CBMs are known to bind to the amorphous regions of cellulose non-competitively and show similar binding affinity towards soluble cello-oligosaccharides. Based on the available crystal structures, these CBMs may display a uni-directional binding preference towards cello-oligosaccharides (based on how the oligosaccharide was bound within the CBM binding cleft). However, molecular dynamics (MD) simulations have indicated no such clear preference. Considering that most soluble oligosaccharides are not always an ideal substrate surrogate to study the binding of CBMs to the native cell wall or cell surface displayed glycans, it is critical to use alternative reagents or substrates. To experimentally assess any binding directionality of CBMs towards soluble cello-oligosaccharides, we have developed a simple solid-state depletion or pull-down binding assay. Here, we specifically orient azido-labeled carbohydrates from the reducing end to alkyne-labeled micron-sized bead surfaces, using click chemistry, to mimic insoluble cell wall surface-displayed glycans. Our results reveal that both family 17 and 28 CBMs displayed a similar binding affinity towards cellohexaose-modified beads, but not cellopentaose-modified beads, which helps rationalize previously reported crystal structure and MD data. This indicates a preferred uni-directional binding of specific CBMs and could explain their co-evolution as tandem constructs appended to endocellulases to increase amorphous cellulose substrate targeting efficiency. Overall, our proposed workflow can be easily translated to measure the affinity of glycan-binding proteins to click-chemistry based immobilized surface-displayed carbohydrates or antigens.

The analysis of particles bound to a surface by flexible tethers can facilitate understanding of various biophysical phenomena (e.g., molecular dynamics of DNA-protein or protein-ligand binding interactions, DNA extensibility and polymer biophysics). Being able to model such systems theoretically can aid in understanding experimentally observed motions and furthermore the limitations of such models can provide insight into modeling complex systems that basic theory sometimes cannot account for. The simulation of tethered particle motion (TPM) allows for efficient analysis of complex behaviors exhibited by such systems, however this type of experiment is rarely taught in undergraduate science classes. We have developed a MATLAB simulation package intended to be used in academic contexts to concisely model and graphically represent the behavior of different tether-particle systems. We show how analysis of the simulation results can be used in biophysical research employing single molecule force spectroscopy (SMFS). Here, our simulation package is capable of modeling any given particle-tether-substrate system and allows the user to generate a parameter space with static and dynamic model components. Our simulation was successfully able to recreate generally observed experimental trends using a recently developed SMFS technique called Acoustic Force Spectroscopy (AFS). Further, the simulation was validated through consideration of the conservation of energy of the tether-bead system, trend analyses, and comparison of particle positional data from actual TPM in silico experiments conducted to simulate data with a parameter space similar to the AFS experimental setup. Overall, our TPM simulator and graphical user interface is suitable for use in an academic context and serves as a template for researchers to set up TPM simulations to mimic their specific SMFS experimental setup.

To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN).Significance StatementCellulases are multi-modular enzymes produced by numerous microbes that catalyze cellulose hydrolysis into glucose. These enzymes play an important role in global carbon cycling as well as cellulosic biofuels production. CBMs are essential components of cellulolytic enzymes involved in facilitating hydrolysis of polysaccharides by tethered catalytic domains (CD). The subtle interplay between CBM binding and CD activity is poorly understood particularly for heterogeneous reactions at solid-liquid interfaces. Here, we report a highly multiplexed single-molecule force spectroscopy method to study CBM dissociation from cellulose to infer the molecular mechanism governing substrate recognition and dissociation. This approach can be broadly applied to study multivalent protein-polysaccharide binding interactions relevant to other carbohydrates such as starch, chitin, or hyaluronan to engineer efficient biocatalysts.

Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have non-catalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two well-studied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond ‘rupture’ assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the ‘cautious’ use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods.Significance StatementMultimodal biomolecular binding interactions involving carbohydrate polymers (e.g., cellulose, starch, chitin, glycosaminoglycans) are fundamental molecular processes relevant to the recognition, biosynthesis, and degradation of all major terrestrial and aquatic biomass. Protein-carbohydrate binding interactions are also critical to industrial biotechnology operations such as enzymatically-catalyzed bioconversion of starch and lignocellulose into biochemicals like ethanol. However, despite the ubiquitous importance of such interfacial processes, we have a poor molecular-level understanding of protein-polysaccharide binding interactions. Here, we provide a comprehensive experimental and theoretical analysis of bulk ensemble versus single-molecule binding interactions of enzyme motors and associated non-catalytic binding domains with cellulosic polysaccharides to highlight the critical limitations of applying classical biochemical assay techniques alone to understanding protein adsorption or biological activity at solid-liquid interfaces.

Down-cycle raw material utilization is a need when it comes to the step out of fossil fuel utilization. Carboxylic acid serve as bulk chemicals, and isolation from industrial aqueous process streams is an ongoing research. Therefore acetic acid esterification of an aqueous acid feed using n-octanol in combination with liquid/liquid-extraction with the solvent mixture n-octanol/n-undecane was investigated. Due to emulsion formation when 4-dodecylbenzenesulfonic acid was used as homogeneous catalyst, the reaction was transferred into a membrane reactor. The homogeneous esterification in the membrane reactor is described by phase transfer catalysis. The combination of acetic acid extraction from aqueous mixture combined with heterogeneous catalysis was investigated in the membrane reactor too. Physical extraction and heterogeneous catalysis lead to conversion of acetic acid, while the reactive solvent Tri-n-octylamine inhibited heterogeneous catalysis by Amberlyst®15. The work provides a novel approach for process intensification of liquid/liquid extraction in combination with an esterification reaction using a membrane reactor.

The urge to achieve climate protection goals and a more prevalent interest in finding alternatives to fossil fuel based products focus attention increasingly on cascade raw material utilization respectively to intensify commercial production processes. The present project provides a study on the combination of liquid/liquid extraction and esterification using the system acetic acid/octanol and 4-dodecylbenzenesulfonic acid as catalyst. The surfactant 4-dodecylbenzenesulfonic acid causes emulsification during extraction, but emulsification was successfully avoided by using supported liquid membrane permeation equipment.

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM-cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1-cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.

With the computational power of modern smartphones constantly increasing, resource intensive applications are becoming feasible to an ever growing extent. In this paper, we report on a research project recently started. Its aim is to develop an application for smartphones that combines pedestrian and public transport navigation including the computation of routes consisting of pedestrian routes and public transport trips and intuitive user guidance at any time of the trip. In particular, we focus on intuitive user guidance based on (LMs) in the surroundings of the user. For this reason, we use collaborative approaches to collect LMs and data about them.

The study aimed to evaluate the test-retest reliability of a newly developed 356 Soccer Shooting Test (356-SST), and the discriminative ability of this test with respect to the soccer players' proficiency level and leg dominance. Sixty-six male soccer players, divided into three groups based on their proficiency level (amateur, n = 24 ; novice semi-professional, n = 18 ; and experienced semi-professional players, n = 24), performed 10 kicks following a two-step run up. Forty-eight of them repeated the test on a separate day. The following shooting variables were derived: ball velocity (BV ; measured via radar gun), shooting accuracy (SA ; average distance from the ball- entry point to the goal centre), and shooting quality (SQ ; shooting accuracy divided by the time elapsed from hitting the ball to the point of entry). No systematic bias was evident in the selected shooting variables (SA: 1.98±0.65 vs. 2.00±0.63 m ; BV: 24.6±2.3 vs. 24.5±1.9 m s-1 ; SQ: 2.92±1.0 vs. 2.93±1.0 m s-1 ; all p>0.05). The intra-class correlation coefficients were high (ICC = 0.70-0.88), and the coefficients of variation were low (CV = 5.3-5.4%). Finally, all three 356-SST variables identify, with adequate sensitivity, differences in soccer shooting ability with respect to the players' proficiency and leg dominance. The results suggest that the 356-SST is a reliable and sensitive test of specific shooting ability in men's soccer. Future studies should test the validity of these findings in a fatigued state, as well as in other populations.

PURPOSE: The aim of the present study was to examine the acute effects of graded physiological strain on soccer kicking performance. METHODS: Twenty-eight semi- professional soccer players completed both experimental and control procedure. The experimental protocol incorporated repeated shooting trials combined with a progressive discontinuous maximal shuttle-run intervention. The initial running velocity was 8 km/h and increasing for 1 km/h every 3 min until exhaustion. The control protocol comprised only eight subsequent shooting trials. The soccer- specific kicking accuracy (KA ; average distance from the ball-entry point to the goal center), kicking velocity (KV), and kicking quality (KQ ; kicking accuracy divided by the time elapsed from hitting the ball to the point of entry) were evaluated via reproducible and valid test over five individually determined exercise intensity zones. RESULTS: Compared with baseline or exercise at intensities below the second lactate threshold (LT2), physiological exertion above the LT2 (blood lactate > 4 mmol/L) resulted in meaningful decrease in KA (11-13% ; p < 0.05), KV (3-4% ; p < 0.05), and overall KQ (13-15% ; p < 0.01). The light and moderate- intensity exercise below the LT2 had no significant effect on soccer kicking performance. CONCLUSIONS: The results suggest that high-intensity physiological exertion above the player's LT2 impairs soccer kicking performance. In contrast, light to moderate physiological stress appears to be neither harmful nor beneficial for kicking performance.

Th epapaer deals with the construction of the shooting test in soccer in regard to fatigue.