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Introduction: Chronic stress is a condition characterized by prolonged stimulation, leading to mental and physical weakness. It can have detrimental effects on individuals' mental health and cognitive function, potentially causing various health issues. This article explores the potential of non-invasive neuromodulation techniques, specifically transcranial direct current stimulation (tDCS) and transcutaneous auricular vagus nerve stimulation (taVNS), in managing chronic stress and improving sleep quality., Methods: The study conducted a randomized, double-blinded, controlled trial with participants experiencing chronic stress. In total, 100 participants were randomly assigned to one of four conditions: the anodal tDCS group ( n = 50), the sham tDCS group ( n = 50), the taVNS group ( n = 50), or the sham taVNS group ( n = 50). Within each condition, participants received five sessions of either active treatment or sham treatment, with 20 min of tDCS over the dorsolateral prefrontal cortex (2 mA) for the tDCS groups, or taVNS on the left ear (20 Hz) for the taVNS groups. At baseline, post-intervention, and 4 weeks thereafter, we evaluated stress using the Lipp's Inventory of Stress Symptoms for Adults (LSSI), perceived stress through the Perceived Stress Scale (PSS-10), and sleep quality via the Pittsburgh Sleep Quality Index (PSQI)., Results: The tDCS and taVNS interventions resulted in reduced stress levels, improved sleep quality, and enhanced perception of stress., Discussion: These findings suggest that tDCS and taVNS hold promise as effective treatments for chronic stress, offering a safe and accessible approach to improving individuals' wellbeing and overall quality of life., Clinical Trial Registration: https://ensaiosclinicos.gov.br/rg/RBR-2ww2ts8, identifier UTN: U1111-1296-1810; Brazilian Registry of Clinical Trials (REBEC) RBR-2ww2ts8., 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 © 2024 dos Reis, Pereira Generoso, Pereira, Teixeira Barú, Candido, Maziero Capello, de Castro, Cardoso, Scoz, Ferreira, da Silva and da Silva.)

Introduction: Chronic anxiety is a statemarked by sustained activation of the masseter muscle, manifesting in both mental and physical strain. This prolonged tension can significantly impact mental wellbeing and cognitive abilities, posing a risk for a range of health complications. This double-blind, randomized, controlled clinical trial investigated the impact of transcutaneous auricular vagus nerve stimulation (TAVNS) on masseter muscle activity, pressure pain threshold (PPT), and anxiety levels in university students with elevated anxiety., Methods: Forty-two participants meeting inclusion criteria were randomly assigned to either active TAVNS or sham TAVNS groups. Various parameters, including masseter muscle electromyographic (EMG) signals, PPT, and Beck Anxiety Inventory (BAI) scores, were assessed before pretreatment, immediately after the intervention week, and 2 weeks follow-up., Results: Active TAVNS significantly reduced both left and right masseter activation during resting mandibular position, persisting for 2 weeks post-intervention. Additionally, TAVNS induced a lasting decrease in both left and right masseter PPT, indicative of altered pain perception. Notably, BAI scores showed a substantial reduction, emphasizing TAVNS as a potential intervention for anxiety, with effects maintained at the 2-week follow-up., Discussion: This study provides comprehensive insights into the multifaceted effects of TAVNS on physiological and psychological aspects associated with anxiety in university students. The promising results underscore TAVNS as a potential neuromodulatory intervention for anxiety-related conditions, warranting further research and clinical exploration., Clinical Trial Registration: https://ensaiosclinicos.gov.br/rg/RBR-4s4kt2r., 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 © 2024 Ferreira, Brites, Fraião, Pereira, Fernandes, de Brito, Pereira Generoso, Maziero Capello, Pereira, Scoz, Silva and Silva.)

The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions., Competing Interests: Conflict of interest The Co-author Carlos M.G.A. Fontes is Founder and CSO of Nzytech Lda, which has manufactured some of the reagents used in this work., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The purpose of this study is to analyze the long-term riders' subjective responses to a standardized bikefitting method on their bicycles. Eighty-six amateur mountain bikers had their riding posture and bicycle components ergonomically adjusted through a 3D kinematic bikefitting method. Validated subjective scales (Feeling, OMNI, and Numerical Rating Pain Scale) were used to assess their overall riding comfort and fatigue along with localized pain for six body parts. Data were collected just before intervention (baseline or pre), immediately after (or post), and 30, 60, 90, and 120 days after the bikefit session. A Student's t -test comparing before bikefit and after 120 days showed significant ( p < 0.05) reduction in localized pain for all six body parts and riding comfort along with a large effect size effect ( d = 1.18) for riding comfort. Although initially reduced, fatigue scores gradually increased over the months, showing a high correlation (r = 0.946) with increased monthly training volume. In conclusion, overall riding discomfort and pain were significantly decreased after a standardized kinematic bikefit session even after 120 days post intervention. However, fatigue scores began to rise after 30 days, showing a high correlation with increasing monthly training volume.

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

Isokinetic tests have been highly valuable to athletic analysis, but their cost and technical operation turn them inaccessible. The purpose of this study was to verify the correlation between unilateral countermovement jump variables and isokinetic data. Thirty-two male professional soccer players were subjected to the isokinetic testing of both knee extensors and flexors in concentric and eccentric muscle contractions. They also executed unilateral countermovement vertical jumps (UCMJ) to compare maximum height, ground reaction force, and impulse power with isokinetic peak torque. Data analysis was conducted through Pearson correlation and linear regression. A high correlation was found between dominant unilateral extensor concentric peak torque and the UCMJ maximum height of the dominant leg. The non-dominant leg jump showed a moderate correlation. No other variable showed statistical significance. Linear regression allowed the generation of two formulae to estimate the peak torque from UCMJ for dominant and non-dominant legs. Although few studies were found to compare our results, leading to more studies being needed, a better understanding of the unilateral countermovement jump may be used in the future as a substitute to the expensive and technically demanding isokinetic testing when it is unavailable, allowing the assessment of lower limb physical asymmetries in athletic or rehabilitation environments.

Objective: To analyse rider's subjective responses after a standardised bicycle ergonomic adjustment method., Methods: Experimental study of 160 healthy, amateur mountain bikers analysed previously and 30 days after a bike-fitting session. The main outcome measures were subjective comfort level (Feeling Scale, FEEL), fatigue (OMINI Scale) and pain (Visual Analogue Scale, VAS)., Results: All variables demonstrated statistical significance between groups pre and post bike-fit session (p<0001). FEEL, OMNI and VAS-knee demonstrated large effect sizes (d=1.30; d=1.39 and d=0.86, respectively). VAS-hands, VAS-neck and VAS-back indicated moderate effect size (d=0.58; d=0.52 and d=0.43, respectively). VAS-groin and VAS-ankle indicated a small size effect (d=0.46 and d=0.43, respectively)., Conclusions: Overall discomfort, fatigue and pain in healthy mountain biker adults improved according to all three scales. The major improvements in pain levels were detected on the knee, hands, back and neck compared with presession values. Groin and ankle pain had smaller improvements but were still significant. Future clinical trials should address the bias effects of this experimental study., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Kinematic analysis aimed toward scientific investigation or professional purposes is commonly unaffordable and complex to use., Objective: The purpose of this study was to verify concurrent validation between a cycling-specific 3D camera and the gold-standard 3D general camera systems., Methods: Overall, 11 healthy amateur male triathletes were filmed riding their bicycles with Vicon 3D cameras and the Retul 3D cameras for bike fitting analysis simultaneously. All 18 kinematic measurements given by the bike fitting system were compared with the same data given by Vicon cameras through Pearson correlation ( r ), intraclass correlation coefficients (ICC), standard error measurements (SEM), and Bland-Altman (BA) analysis. Confidence intervals of 95% are given., Results: A very high correlation between cameras was found on six of 18 measurements. All other presented a high correlation between cameras (between 0.7 and 0.9). In total, six variables indicate a SEM of less than one degree between systems. Only two variables indicate a SEM higher than two degrees between camera systems. Overall, four measures indicate bias tendency according to BA., Conclusions: The cycling-specific led-emitting 3D camera system tested revealed a high or very high degree of correlation with the gold-standard 3D camera system used in laboratory motion capture. In total, 14 measurements of this equipment could be used in sports medicine clinical practice and even by researchers of cycling studies.

Background/aim: The purpose of this study was to compare the isokinetic peak torque profiles from the quadriceps and hamstrings muscles during concentric and eccentric contractions in elite Brazilian soccer players across different field positions and age categories. Our hypothesis was that soccer players from different field positions are subjected to different ageing-related effects on their isokinetic peak torque., Methods: This is a retrospective study based on professional elite-level soccer players between the years 2009 and 2019. It included 570 adult males who played for at least 5 years on first or second Brazilian divisions. Playing positions were divided as: goalkeepers, defenders, sidebacks, midfielders and forwards. Age categories were also divided as: G1 (17-20 years old), G2 (21-24 years old), G3 (25-28 years old), G4 (29-32 years old) and G5 (33 years old or more)., Results: The results indicate a moderate effect of age (F(4545)=8.197; p<0.001; η 2 =0.057) and a small effect of playing position (F(4545)=2.993; p<0.05; η 2 =0.021) on torque of concentric extensors; mainly from midfielders and goalkeepers with 29 years or more., Conclusions: Soccer players from different field positions are subjected to different ageing related effects on their muscular performance during their career special attention should be given to these players to avoid reduction in physical performance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin-dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

It is now well established that exogenous β-1,4-xylanases improve the nutritive value of wheat-based diets for poultry. Among other factors, the mechanism of action of exogenous enzymes may involve a microbial route resulting from the generation of prebiotic xylo-oligosaccharides (XOS) in the birds' gastro-intestinal (GI) tract. In a series of three experiments, the effect of XOS on the performance of broilers fed wheat or corn-based diets was investigated. In experiment 1, birds receiving diets supplemented with XOS displayed an increased weight gain (P = 0.08). The capacity of XOS to improve the performance of animals during a longer trial (42 d) was investigated (Experiment 2). The data revealed that diet supplementation with XOS, tested at two incorporation rates (0.1 and 1 g/kg), or with an exogenous β-1,4-xylanase resulted in an increased nutritive value of the wheat-based diet. An improvement in animal performance was accompanied by a shift in the microbial populations colonizing the upper portions of the GI tract. XOS were also able to improve the performance of broilers fed a corn-based diet, although the effects were not apparent at incorporation rates of 10 g/kg. Together these studies suggest that in some cases the capacity of β-1,4-xylanases to improve the nutritive value of wheat-based diets is more related to their ability to produce prebiotic XOS than to their ability to degrade arabinoxylans. The extremely low quantities of XOS used in this study also challenge the depiction of a prebiotic being a quantitatively fermented substrate. These data also bring into question the validity of the "cell wall" mechanism, as XOS elicited an effect with clearly no action on endosperm cell wall integrity and yet the performance effects noted were equivalent or superior to the added enzymes.

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

1. Endo-1,4-β-xylanase is known to improve the nutritive value of wheat-based diets for poultry by degrading dietary arabinoxylans. However, broilers' response to supplementation of wheat-based diets with exogenous endo-1,4-β-xylanase is not always observed. 2. In this study, 108 different wheat lots were analysed for levels of extract viscosity as well as for endogenous endo-1,4-β-xylanase activity, and the impact of these two variables in animal performance was tested. 3. Results revealed that endogenous endo-1,4-β-xylanase activity and extract viscosity content varied widely among different wheat lots. Thus, a trial was conducted to evaluate the efficacy of exogenous enzyme supplementation in broiler diets using wheats with different levels of extract viscosity and endogenous endo-1,4-β-xylanase activity. 4. The data revealed that exogenous enzyme supplementation was only effective when the wheat present in the diet had high levels of extract viscosity (14.8 cP) with low endogenous endo-1,4-β-xylanase activity (347.0 U/kg). Nevertheless, it is apparent that exogenous microbial xylanases reduce digesta extract viscosity and feed conversion ratio independently of the endogenous properties presented by different wheat lots. 5. The data suggest that extract viscosity and/or endogenous endo-1,4-β-xylanase activity affect the response to enzyme supplementation by poultry fed on wheat-based diets.

The cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type-I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaA-enzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type-I cohesin-dockerin specificity in A. cellulolyticus Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA- and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both, of the ligand-binding sites of ScaB, whereas attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Abtract: Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens' cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambdaZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M(r) of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M(r) of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.