Oriented display of cello-oligosaccharides for pull-down binding assays to distinguish binding preferences of glycan binding proteins.

The production of biofuels from lignocellulosic biomass using carbohydrate-active enzymes like cellulases is key to a 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 better understand the binding of type B CBMs towards smaller 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 may indicate 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.

Acoustic force spectroscopy reveals subtle differences in cellulose unbinding behavior of carbohydrate-binding modules.

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.

Does a Physical Activity Intervention on Classroom-Based Ergometers During Teaching Lessons Effect Physical Fitness, Body Composition, and Health-Related Blood Parameters? A Pilot Cluster Randomized Controlled Study.

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 < .001), maximal aerobic power (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.

Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III.

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.

Reliability and Discriminative Ability of a New Method for Soccer Kicking Evaluation.

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.

The acute effects of graded physiological strain on soccer kicking performance: a randomized, controlled cross-over study.

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.