COSMIC-dFBA: A novel multi-scale hybrid framework for bioprocess modeling.

Metabolism governs cell performance in biomanufacturing, as it fuels growth and productivity. However, even in well-controlled culture systems, metabolism is dynamic, with shifting objectives and resources, thus limiting the predictive capability of mechanistic models for process design and optimization. Here, we present Cellular Objectives and State Modulation In bioreaCtors (COSMIC)-dFBA, a hybrid multi-scale modeling paradigm that accurately predicts cell density, antibody titer, and bioreactor metabolite concentration profiles. Using machine-learning, COSMIC-dFBA decomposes the instantaneous metabolite uptake and secretion rates in a bioreactor into weighted contributions from each cell state (growth or antibody-producing state) and integrates these with a genome-scale metabolic model. A major strength of COSMIC-dFBA is that it can be parameterized with only metabolite concentrations from spent media, although constraining the metabolic model with other omics data can further improve its capabilities. Using COSMIC-dFBA, we can predict the final cell density and antibody titer to within 10% of the measured data, and compared to a standard dFBA model, we found the framework showed a 90% and 72% improvement in cell density and antibody titer prediction, respectively. Thus, we demonstrate our hybrid modeling framework effectively captures cellular metabolism and expands the applicability of dFBA to model the dynamic conditions in a bioreactor.

Guidelines for extracting biologically relevant context-specific metabolic models using gene expression data.

Genome-scale metabolic models comprehensively describe an organism's metabolism and can be tailored using omics data to model condition-specific physiology. The quality of context-specific models is impacted by (i) choice of algorithm and parameters and (ii) alternate context-specific models that equally explain the -omics data. Here we quantify the influence of alternate optima on microbial and mammalian model extraction using GIMME, iMAT, MBA, and mCADRE. We find that metabolic tasks defining an organism's phenotype must be explicitly and quantitatively protected. The scope of alternate models is strongly influenced by algorithm choice and the topological properties of the parent genome-scale model with fatty acid metabolism and intracellular metabolite transport contributing much to alternate solutions in all models. mCADRE extracted the most reproducible context-specific models and models generated using MBA had the most alternate solutions. There were fewer qualitatively different solutions generated by GIMME in E. coli, but these increased substantially in the mammalian models. Screening ensembles using a receiver operating characteristic plot identified the best-performing models. A comprehensive evaluation of models extracted using combinations of extraction methods and expression thresholds revealed that GIMME generated the best-performing models in E. coli, whereas mCADRE is better suited for complex mammalian models. These findings suggest guidelines for benchmarking -omics integration algorithms and motivate the development of a systematic workflow to enumerate alternate models and extract biologically relevant context-specific models.

Phenotype-centric modeling for rational metabolic engineering.

Phenotype-centric modeling enables a paradigm shift in the analysis of mechanistic models. It brings the focus to a network's biochemical phenotypes and their relationship with measurable traits (e.g., product yields, system dynamics, signal amplification factors, etc.) and away from computationally intensive simulation-centric modeling. Here, we explore applications of this new modeling strategy in the field of rational metabolic engineering using the amorphadiene biosynthetic network as a case study. This network has previously been studied using a mechanistic model and the simulation-centric strategy, and thus provides an excellent means to compare and contrast results obtained from these two very different strategies. We show that the phenotype-centric strategy, without values for the parameters, not only identifies beneficial intervention strategies obtained with the simulation-centric strategy, but it also provides an understanding of the mechanistic context for the validity of these predictions. Additionally, we propose a set of hypothetical strains with the potential to outperform reported production strains and to enhance the mechanistic understanding of the amorphadiene biosynthetic network. Further, we identify the landscape of possible intervention strategies for the given model. We believe that phenotype-centric modeling can advance the field of rational metabolic engineering by enabling the development of next generation kinetics-based algorithms and methods that do not rely on a priori knowledge of kinetic parameters but allow a structured, global analysis of system design in the parameter space.

Individualization of Pinus radiata Canopy from 3D UAV Dense Point Clouds Using Color Vegetation Indices.

The location of trees and the individualization of their canopies are important parameters to estimate diameter, height, and biomass, among other variables. The very high spatial resolution of UAV imagery supports these processes. A dense 3D point cloud is generated from RGB UAV images, which is used to obtain a digital elevation model (DEM). From this DEM, a canopy height model (CHM) is derived for individual tree identification. Although the results are satisfactory, the quality of this detection is reduced if the working area has a high density of vegetation. The objective of this study was to evaluate the use of color vegetation indices (CVI) in canopy individualization processes of Pinus radiata. UAV flights were carried out, and a 3D dense point cloud and an orthomosaic were obtained. Then, a CVI was applied to 3D point cloud to differentiate between vegetation and nonvegetation classes to obtain a DEM and a CHM. Subsequently, an automatic crown identification procedure was applied to the CHM. The results were evaluated by contrasting them with results of manual individual tree identification on the UAV orthomosaic and those obtained by applying a progressive triangulated irregular network to the 3D point cloud. The results obtained indicate that the color information of 3D point clouds is an alternative to support individualizing trees under conditions of high-density vegetation.

Impact of elite soccer coaching change on team performance according to coach- and club-related variables.

A coaching change is an extreme, but frequently occurring phenomenon in elite soccer with its impact on team success debatable. The aim of the current study was twofold: (i) to compare team's performance when coached by new and old coaches; and (ii) to investigate the impact of a coaching change on team's performance according to coach- and club-related factors. All in-season coaching changes from the 2010-11 to 2017-18 seasons within the Spanish, French, English, German and Italian professional leagues were examined. Team performance was assessed as points awarded from match outcome over 1-20 matches prior to and following the coaching change. Four independent variables (coach's experience, team's budget, whether the coach had been an elite former player or not, and whether the coach was a novice or not) were included into linear regression modelling. The main results showed that team's short-term performance was improved significantly with a change to a new coach with this impact declining in the longer term (> 10 matches). Specifically, the number of points (1.15-1.32 vs. 0.37-1.03, p < 0.05) and the moving average of points (1.19-1.31 vs. 0.37-1.04, p < 0.05) awarded per match were significantly greater after the coaching change. Further, the winning effect due to the new coach was independent of coach-related factors such as coaching experience or the new coach being a former elite player. A critical organisational decision to change coaches may provide an essential stimulus for future team success in elite soccer.

Ibuprofen removal by a microfiltration membrane bioreactor during the startup phase.

The behavior of ibuprofen (IBU) during the startup phase of a microfiltration membrane bioreactor (MBR) was determined. A full-scale experimental installation treating real urban wastewater was used for the study. The MBR was composed of an anoxic and an aerobic bioreactors working in pre-denitrification configuration, followed of a membrane reactor. A full mass balance was carried out to estimate the contribution of biotransformation and sorption to biomass to the overall removal of the IBU. During the startup phase of the MBR system there were significant oscillations of the operational variables, mainly of the sludge retention time (SRT); nevertheless, the capacity of the system for IBU removal was very high, with yields of over 94%, despite reaching minimum SRT values of 4.15 d. The main IBU removal occurs in the aerobic reactor, both in the liquid phase and the one associated with the sludge, while in the anoxic bioreactor the removal was scarce, although a certain transfer of IBU from the liquid phase to the sludge took place under anoxic conditions. Despite the high IBU removal yields during startup, the SRT was the most influential variable in IBU removal, an effect observed in all bioreactors, particularly in the anoxic one.

Goal effectiveness after players' dismissals in professional futsal teams.

The purpose of this study was to analyse the effect of players' dismissals on the outcome of attacks in elite futsal matches, and to establish the performance profile of the attacks made in numerical superiority by elite futsal teams. One hundred and twenty five attacking game situations in numerical superiority (dismissal of opponents from defensive team) were analysed from the regular season of the Spanish professional Futsal League. The effect of contextual-related variables (quality of opposition, match-location, match-periods, opponent team's fouls, match-status, attack-duration and match-type) on goal effectiveness was analysed using binomial logistic regression and two-step cluster analysis. Results from the binary logistic regression showed that the highest attack effectiveness was achieved when the teams play at home, perform the attack during minutes 33-36 and the opposing team has 3 fouls. Secondly, the two-step cluster analysis technique allowed identifying four types of attacks when the teams were playing with numerical superiority. The results showed the great importance (in order) of match-type, match-status, attacking team's fouls, match-period, quality of opposition, opposing team's fouls, match-location, goal situation, and attack duration. The identified trends may help coaches to design the superiority/inferiority scenarios more specifically during training and to monitor them during competition.

Technical and physical match performance of teams in the 2018 FIFA World Cup: Effects of two different playing styles.

In order to investigate the effect of playing styles on the match performance of football teams, data were analysed on 18 technical performance-related variables and 8 physical performance-related variables from 59 matches in the 2018 FIFA World Cup. A k-means cluster analysis was conducted to classify all match observations into two clusters of tactical approach in order to identify the playing styles of teams (characterised as direct-play, possession-play and mixed-play). Separate Poisson regression models were run in the generalised mixed linear modelling to examine the differences in technical and physical performance between teams classified as using different playing styles when facing different opponents. Results showed that possession-play characterised teams achieved higher values in all the variables related to goal scoring, attacking and passing (ES: 0.32 ~ 1.27) and covered more distance in sprints and high-intensity running (ES: 0.33 ~ 0.47) than direct-play characterised teams. Both possession- and direct-play characterised teams achieved higher values in passing, pass accuracy and delivery into the attacking third playing against direct-play characterised teams than playing against possession-play characterised sides (ES: 0.22 ~ 0.98). These findings may provide insights into the establishment of performance profiles of teams with different tactical styles and the development of specific training drills to optimise playing style.

Relationships Among Circuit Training, Small-Sided and Mini Goal Games, and Competition in Professional Soccer Players: A Comparison of On-Field Integrated Training Routines.

Giménez, JV and Gomez, MA. Relationships among circuit training, small-sided and mini goal games, and competition in professional soccer players: A comparison of on-field integrated training routines. J Strength Cond Res 33(7): 1887-1896, 2019-The aim of this study was to investigate and compare different physical variables and load indicators of 2 small-sided game (SSG) formats and ball circuit training (CT). Fourteen professional players participated in 3 training routines using a similar occupied area per player (90 m). The CT, SSGs, and mini goal games (MGs) consisted of 8 repetitions of 4-minute game play, interspersed by 2 minutes of active recovery, and data were compared with the first 32 minutes of 2 competitive match simulations (MS). All movement patterns from walking to sprinting were recorded using 10-Hz global positioning system devices, whereas player perception of exertion was recorded after trial using a visual analogue scale. Practical differences among the 3 drills and MS were analyzed using magnitude-based inferences. The results suggested that the training routines did not exactly replicate the movement patterns of a competitive match. Furthermore, this study provides evidence that if high-intensity play is preferred, then SSGs should be emphasized (because they provide more total accelerations compared with the other drills; most likely effects). Moreover, the CT showed lower load and distance covered (m) than the MGs and SSGs. In conclusion, these drills may be useful for competition and impact microcycles (i.e., intermittent efforts with accelerations, decelerations, and walking actions) to achieve the specific adaptations of high-intensity efforts.

Effects of water re-allocation in the Ebro river basin: A multiregional input-output and geographical analysis.

The quality and availability of water are affected by numerous variables, through which the evaluation of water uses from different perspectives, and policy proposals to save water have now become essential. This paper aims to study water use and the water footprint from a river basin perspective, taking into account regions, sectors, and municipalities, while considering the physical frontier along with the administrative sectors. To this end, we have constructed a multi-regional input-output table for the Ebro river basin, disaggregating the primary sector into 18 different crops and 6 livestock groups. We pay special attention to crop production because it is the most water-consuming industry. The construction of the multi-regional input-output model represents an important contribution to the literature, in itself, since, to the best of our knowledge, it is the first to be built for this large basin. We extend this multi-regional input-output model to assess the water footprint by sectors and regions within the basin. We use these data to propose two scenarios: reallocating final demand to reduce the blue water footprint (scenario 1), and increasing value added (scenario 2). These scenarios outline the opportunity costs of saving water in socioeconomic terms in the basin. In another application, we downscale the multi-regional input-output model results at the municipal level and depict them using a geographical information system, identifying the hotspots and the areas that would pay for the socioeconomic opportunity costs of saving water. Our results suggest that saving 1 hm3 of blue water could cost around €41,500 of value added if we consider the entire basin. However, this water re-allocation implies losses and gains at the municipal level: some municipalities would reduce value added by more than €30,000, while others would gain more than €85,000 of value added. These tools and results can be useful for policy makers when considering re-allocating water. The contribution and the novelty of this paper is the construction of the multiregional input-output model for the Ebro river basin, and its link with geographical systems analysis at the municipal level.

Phenotype-centric modeling for elucidation of biological design principles.

A recently developed 'phenotype-centric' modeling strategy combines four innovations with the potential to advance our understanding of complex biological systems: (1) a rigorous mathematical definition of biochemical phenotypes, (2) a method for enumerating the phenotypic repertoire based on the biomolecular network architecture, (3) an integrated suite of computational algorithms for the efficient prediction of parameter values and analysis of the phenotypic repertoire, and (4) a user-focused environment for navigating the resulting space of phenotypes and identifying biologically relevant features and system design principles. These innovations will facilitate deterministic and stochastic simulations that require parameter values, will accelerate both hypothesis discrimination in systems biology and the design cycle in synthetic biology. Here we first review the fundamental definition of biochemical phenotype that enables this new modeling strategy and give an overview of the strategy using a simple system from phage λ to provide an example of a global design principle. Second, we illustrate this approach in more detail with an application to a common network architecture involving positive and negative feedback. We report system design principles related to the global tolerances of this system's phenotypes. Finally, we apply the phenotype-centric strategy to a logic network and compare the results with those obtained from a Boolean approach. Mechanistic and Boolean models have well-documented complementary advantages and disadvantages. Mechanistic models have the advantage of being biologically realistic; however, they also are limited by the large number of kinetic parameters whose values are largely unknown. Boolean models have the advantage of being parameter free; however, they also are limited by the absence of well-known physical and chemical constraints. We show that the phenotype-centric modeling strategy combines advantages of both.

Importance of the knowledge of pathological processes for risk-based inspection in pig slaughterhouses (Study of 2002 to 2016).

OBJECTIVE: The objective of this work was to determine the prevalence of the pathologies that caused the condemnation of pig carcasses in an area of intensive pig farming and Mediterranean climatology and to evaluate their influence in a risk-based inspection procedure for slaughterhouses. METHODS: A retrospective observational investigation was carried out from 2002 to 2016 into the pathological processes that caused the condemnation of pig carcasses in a slaughterhouse from South-eastern Spain. The seasonal effect on the causes of condemnation carcass was reported. Negative binomial model was used to evaluate the effect of season on the rate of antemortem rejections and post-mortem condemnations. Histopathological examinations were performed to confirm the diagnosis. RESULTS: The risk of antemortem rejections (0.0564%) was significantly greater in summer (risk ratio [RR] = 1.57). Autumn was associated with higher rate (RR = 1.69) of the total postmortem condemnations (0.1046%). Significantly higher rates of pronounced anaemia (0.0111%) were observed in summer (RR = 3.20). The main causes of anaemia were observed gastroesophageal ulcers and haemorrhagic enteropathies. Significantly highest risk of erysipelas (0.0074%) were observed in autumn (RR = 5.485). About other zoonosis, only eight cases (0.0013%) of carcasses were declared unfit due to tuberculosis lesions. Porcine muscular cysticercosis was not detected. Nevertheless, nonspecific causes such as generalized infections and emaciation represented the half of the condemned carcasses (50.90%). CONCLUSION: The pathologies leading to the condemnation of carcasses in this study can be considered representative of the pathologies that affect the pig population from a region with a high intensive production and Mediterranean climatology because this slaughterhouse receives a lot of animals from many farms of different size in a high intensive pig production zone (Mediterranean region). Increased knowledge of environmental factors that may foment the appearance of the diseases is essential for implementing inspection programs based on risk assessment in pig's slaughterhouses.

Physical responses of professional soccer players during 4 vs. 4 small-sided games with mini-goals according to rule changes.

The aim of this study was to investigate the influence of the number of ball touches authorised per game (one touch [T1], two touches [T2], and free touches [FT]) on the players' physical responses throughout the bouts in 4 vs 4 soccer small-sided games (SSGs) with mini-goals (without a goalkeeper). Fourteen professional Polish players (age 23.2±2.7 years, height 177.9±6.1 cm, weight: 73.2±6.9 kg, body fat 12.6±2%, playing experience: 14±5 years) completed nine series of 4 vs 4 SSGs. Each trial included three series of SSGs with a game duration of 4 minutes on an equal sized pitch (30x24 m; 720 m2; individual occupied area per player=90 m2). Differences in physical responses and time-motion characteristics of players were measured with the Global Positioning System (GPS) and assessed using a repeated measures ANOVA to compare the three game conditions and the magnitude-based inference to evaluate the pairwise comparison effects. The results showed that only the variables distance covered at low speed, time walking, time at low speed, and accelerations of >4 m/s² were statistically significantly different among game conditions. The pairwise comparisons only identified significant effects for distance covered at low speed (between FT and T2), for time walking (between FT and T1), for time at moderate and low speed (between FT and T2), and for accelerations of >4 m/s² (between FT and T1). The players' performances are affected by the ball touch constraint during SSGs with mini-goals. The results provide useful information for training and task design that replicate specific physical demands (i.e., accelerations of >4 m/s², time walking or running at a lower speed).

Behaviour of biopolymeric substances in the activated sludge of an MBR system working with high hydraulic retention time.

This study was undertaken to analyse the activated sludge of a membrane bioreactor (MBR), the behaviour of extracellular polymeric substances (EPS) and soluble microbial products (SMP) as well as their biopolymers composition, in the activated sludge of a membrane bioreactor (MBR) and their influence on membrane fouling were analysed. For the experiment an experimental fullscale MBR working with real urban wastewater at high hydraulic retention time with a variable sludge-retention time (SRT) was used. The MBR system worked in denitrification/nitrification conformation at a constant flow rate (Q = 0.45 m3/h) with a recirculation flow rate of 4Q. The concentrations of SMP in the activated sludge were lower than the concentrations of EPS over the entire study, with humic substances being the main components of the two biopolymers. SMP and, more specifically, SMP carbohydrates, were the most influential biopolymers in membrane fouling, while for EPS and their components, no relation was found with fouling. The SRT and temperature were the operational variables that most influenced the SMP and EPS concentration, causing the increase of SRT and temperature a lower concentration in both biopolymers, although the effect was not the same for all the components, particularly for the EPS carbohydrates, which increased with longer SRTs. Both operational variables were also the ones most influential on the concentration of organic matter of the effluent, due to their effect on the SMP. The volatile suspended solid/total suspended solid (VSS/TSS) ratio in the activated sludge can be applied as a good indicator of the risk of membrane fouling by biopolymers in MBR systems.

Explaining Match Outcome During The Men's Basketball Tournament at The Olympic Games.

In preparation for the Olympics, there is a limited opportunity for coaches and athletes to interact regularly with team performance indicators providing important guidance to coaches for enhanced match success at the elite level. This study examined the relationship between match outcome and team performance indicators during men's basketball tournaments at the Olympic Games. Twelve team performance indicators were collated from all men's teams and matches during the basketball tournament of the 2004-2016 Olympic Games (n = 156). Linear and non-linear analyses examined the relationship between match outcome and team performance indicator characteristics; namely, binary logistic regression and a conditional interference (CI) classification tree. The most parsimonious logistic regression model retained 'assists', 'defensive rebounds', 'field-goal percentage', 'fouls', 'fouls against', 'steals' and 'turnovers' (delta AIC <0.01; Akaike weight = 0.28) with a classification accuracy of 85.5%. Conversely, four performance indicators were retained with the CI classification tree with an average classification accuracy of 81.4%. However, it was the combination of 'field-goal percentage' and 'defensive rebounds' that provided the greatest probability of winning (93.2%). Match outcome during the men's basketball tournaments at the Olympic Games was identified by a unique combination of performance indicators. Despite the average model accuracy being marginally higher for the logistic regression analysis, the CI classification tree offered a greater practical utility for coaches through its resolution of non-linear phenomena to guide team success.

Impact of tacrolimus and mycophenolate mofetil regimen vs. a conventional therapy with steroids on cardiovascular risk in liver transplant patients.

The aim of this study was to evaluate the impact of a steroid-free regimen with tacrolimus and mycophenolate mofetil (modified therapy) vs. a standard regimen of tacrolimus and steroids on the cardiovascular risk score of liver transplant recipients. Patients who received a liver transplant were randomized to a modified therapy (n = 58) or a standard regimen (n = 59). Both groups were balanced at baseline, except for a higher prevalence of diabetes mellitus (DM) (p < 0.01) and a higher serum creatinine concentration (p < 0.05) in the modified therapy group. After 12 months, the prevalence of new-onset DM, arterial hypertension, hypercholesterolemia, hypertriglyceridemia, and changes in cardiovascular risk factors was similar in both groups. The increase in serum creatinine (mg/dL) compared to baseline at one yr post-transplantation was numerically lower in the modified therapy group (0.22 ± 0.42) than in the standard regimen group (0.41 ± 0.67) (p = 0.068). Although estimated cardiovascular risk score did not vary significantly compared to baseline in either group, there was a slight reduction in the modified regimen (-0.27 ± 2.87) vs. a mild increase (0.17 ± 2.94) in the standard regimen (p = 0.566). In conclusion, a steroid-free regimen with tacrolimus and mycophenolate mofetil was associated with a trend toward better preservation of kidney function and reduction of cardiovascular risk score.

Mesorectal pathologic assessment in two grades predicts accurately recurrence, positive circumferential margin, and correlates with survival.

BACKGROUND AND OBJECTIVES: We aimed to determine the prognostic value of the mesorectum quality assessed in a two-grade system compared with a classic system. METHODS: Consecutive patients undergoing surgery for rectal cancer were included (n = 103). Mesorectum was assessed into three grades (classic system: complete, nearly complete, incomplete) and compared with a two-grade system (adequate, inadequate). RESULTS: Mesorectum was complete in 62 (60.25%) patients, nearly complete in 21, and incomplete in 20. Reassessment showed adequate mesorectum in 83 (80.5%) patients and inadequate in 20. A R0 resection was achieved in 90.4% of adequate mesorectum and in 65% of inadequate mesorectum (P = 0.006). Recurrence was present in 18% of adequate mesorectum patients as compared with 50% of inadequate mesorectum (P = 0.003). The classic system failed to accurately predict the 5-year survival rate between complete (78.9%) and nearly complete (86.2%) categories (P = 0.235); whereas a two grading system showed a 5-year survival rate of 80.8% for adequate versus 39.3% for inadequate (P = 0.034). CONCLUSION: High recurrence occurred in inadecuate mesorectum patients and was correlated with R1/R2 resections, positive margins, and decreased survival. We propose a simplified classification of mesorectum that correlates with survival and overall recurrence.

Integrated in-line coagulation-aerated ultrafiltration for drinking-water production: A case study from laboratory to pilot plant.

A pilot-scale study was made for drinking-water production from low-quality influents with in-line coagulation as pre-treatment of aerated ultrafiltration. Optimum flocculation parameters were previously determined in the laboratory, searching for large and strong flocs that could resist aeration without increasing the membrane fouling. Nevertheless, the comparison of the jar tests and the pilot-scale results showed that the former could help pre-dimensioning the flocculation facilities but could not precisely reproduce the behavior of the flocs in the membrane tank. The optimum coagulant dosage enabled the highest dissolved organic carbon (DOC) removal without affecting membrane fouling. Additionally, as opposed to the jar-test results, long retention times led to the lowest fouling rates while the velocity gradient affected neither the effluent quality nor the membrane fouling. These findings suggested that the influence of the flocculation parameters was masked by aeration inside the membrane tank but, even so, 43% and 82% DOC and UVA254 removals, respectively, were reached. Furthermore, the growth of the total membrane resistance with time was logarithmic instead of linear, confirming the suitability of the pre-treatment for low-quality influents.

Comparison of Home Advantage in College and Professional Team Sports in the United States.

Home advantage in seven American college team sports (baseball, basketball, football, hockey, lacrosse, soccer and women's basketball) was compared with professional leagues in the United States for the same sports and for the same time period. A total of 81,063 college games and 22,477 professional games were analyzed for the four seasons 2006-07 to 2009-10. There was a significant home advantage, as measured by home winning percentage, in all sports, both college and professional. The overall home advantage in college sports was significantly greater than in professional sports (p<0.015). The mean difference was 3.73 home winning percentage points, being greatest for baseball, basketball, and hockey (all p<0.001). Plausible explanations for these results include differences in college and professional competition in terms of familiarity with local conditions, referee bias, territoriality and psychological factors. However, the influence of travel fatigue was inconclusive. Only for soccer was the home advantage greater for professionals. This was the only sport where crowd size appeared to be having an effect. In addition the rules of college soccer allow more substitution and hence greater coach intervention than in professional soccer, a factor that could also be reducing home advantage.

Klotho inversely relates with carotid intima- media thickness in atherosclerotic patients with normal renal function (eGFR ≥60 mL/min/1.73m2): a proof-of-concept study.

Introduction: Klotho protein is predominantly expressed in the kidneys and has also been detected in vascular tissue and peripheral blood circulating cells to a lesser extent. Carotid artery intima-media thickness (CIMT) burden, a marker of subclinical atherosclerosis, has been associated with reductions in circulating Klotho levels in chronic kidney disease patients, who show reduced levels of this protein at all stages of the disease. However, the contribution of serum Klotho and its expression levels in peripheral blood circulating cells and in the carotid artery wall on the CIMT in the absence of kidney impairment has not yet been evaluated. Methods: We conducted a single-center study in 35 atherosclerotic patients with preserved kidney function (eGFR≥60 mL/min/1.73m2) subjected to elective carotid surgery. Serum levels of Klotho and cytokines TNFa, IL6 and IL10 were determined by ELISA and transcripts encoding for Klotho (KL), TNF, IL6 and IL10 from vascular segments were measured by qRT-PCR. Klotho protein expression in the intima-media and adventitia areas was analyzed using immunohistochemistry. Results: APatients with higher values of CIMT showed reduced Klotho levels in serum (430.8 [357.7-592.9] vs. 667.8 [632.5-712.9] pg/mL; p<0.001), mRNA expression in blood circulating cells and carotid artery wall (2.92 [2.06-4.8] vs. 3.69 [2.42-7.13] log.a.u., p=0.015; 0.41 [0.16-0.59] vs. 0.79 [0.37-1.4] log.a.u., p=0.013, respectively) and immunoreactivity in the intimal-medial area of the carotids (4.23 [4.15-4.27] vs. 4.49 [4.28-4.63] log µm2 p=0.008). CIMT was inversely related with Klotho levels in serum (r= -0.717, p<0.001), blood mRNA expression (r=-0.426, p=0.011), and with carotid artery mRNA and immunoreactivity levels (r= -0.45, p=0.07; r= -0.455, p= 0.006, respectively). Multivariate analysis showed that serum Klotho, together with the gene expression levels of tumor necrosis factor TNFa in blood circulating cells, were independent determinants of CIMT values (adjusted R2 = 0.593, p<0.001). Discussion: The results of this study in subjects with eGFR≥60mL/min/1.73m2 show that patients with carotid artery atherosclerosis and higher values of CIMT present reduced soluble Klotho levels, as well as decreased KL mRNA expression in peripheral blood circulating cells and Klotho protein levels in the intima-media of the carotid artery wall.