Activation of coagulation FXI promotes endothelial inflammation and amplifies platelet activation in a nonhuman primate model of hyperlipidemia.

Background: Hyperlipidemia is associated with chronic inflammation and thromboinflammation. This is an underlying cause of several cardiovascular diseases, including atherosclerosis. In diseased blood vessels, rampant thrombin generation results in the initiation of the coagulation cascade, activation of platelets, and endothelial cell dysfunction. Coagulation factor (F) XI represents a promising therapeutic target to reduce thromboinflammation, as it is uniquely positioned at an intersection between inflammation and thrombin generation. Objectives: This study aimed to investigate the role of FXI in promoting platelet and endothelial cell activation in a model of hyperlipidemia. Methods: Nonhuman primates (NHPs) were fed a standard chow diet (lean, n = 6) or a high-fat diet (obese, n = 8) to establish a model of hyperlipidemia. Obese NHPs were intravenously administered a FXI blocking antibody (2 mg/kg) and studied at baseline and at 1, 7, 14, 21, and 28 days after drug administration. Platelet activation and inflammatory markers were measured using fluorescence-activated cell sorting or enzyme-linked immunosorbent assay. Molecular imaging was used to quantify vascular cell adhesion molecule 1 (VCAM-1) expression at the carotid bifurcation. Results: Obese NHPs demonstrated increased sensitivity for platelet P-selectin expression and phosphatidylserine exposure in response to platelet GPVI or PAR agonists compared with lean NHPs. Obese NHPs exhibited elevated levels of C-reactive protein, cathepsin D, and myeloperoxidase compared with lean NHPs. Following pharmacological inhibition of FIX activation by FXIa, platelet priming for activation by GPVI or PAR agonists, C-reactive protein levels, and endothelial VCAM-1 levels were reduced in obese NHPs. Conclusion: FXI activation promotes the proinflammatory phenotype of hyperlipidemia by priming platelet activation and inciting endothelial cell dysfunction.

Cellular mechanisms of contextual fear memory reconsolidation: Role of hippocampal SFKs, TrkB receptors and GluN2B-containing NMDA receptors.

Memories are stored into long-term representations through a process that depends on protein synthesis. However, a consolidated memory is not static and inflexible and can be reactivated under certain circumstances, the retrieval is able to reactivate memories and destabilize them engaging a process of restabilization known as reconsolidation. Although the molecular mechanisms that mediate fear memory reconsolidation are not entirely known, so here we investigated the molecular mechanisms in the hippocampus involved in contextual fear conditioning memory (CFC) reconsolidation in male Wistar rats. We demonstrated that the blockade of Src family kinases (SFKs), GluN2B-containing NMDA receptors and TrkB receptors (TrkBR) in the CA1 region of the hippocampus immediately after the reactivation session impaired contextual fear memory reconsolidation. These impairments were blocked by the neurotrophin BDNF and the NMDAR agonist, D-Serine. Considering that the study of the link between synaptic proteins is crucial for understanding memory processes, targeting the reconsolidation process may provide new ways of disrupting maladaptive memories, such as those seen in post-traumatic stress disorder. Here we provide new insights into the cellular mechanisms involved in contextual fear memory reconsolidation, demonstrating that SFKs, GluN2B-containing NMDAR, and TrkBR are necessary for the reconsolidation process. Our findings suggest a link between BDNF and SFKs and GluN2B-containing NMDAR as well as a link between NMDAR and SFKs and TrkBR in fear memory reconsolidation. These preliminary pharmacological findings provide new evidence of the mechanisms involved in the reconsolidation of fear memory and have the potential to contribute to the development of treatments for psychiatric disorders involving maladaptive memories.

Relationship between relative age measured through decimal age, physical variables and anthropometry in elite youth soccer players.

INTRODUCTION: Age differences between athletes born in the same year, as well as an over-representation of older players, are known as the Relative Age Effect (RAE). Players born at the beginning of the selection year have a physical and anthropometric advantage over their younger peers. Experts keep looking for new prediction variables for talent identification. OBJECTIVES: The aim of the study is to correlate anthropometric, strength and power variables with the relative age (RA) and the level of the teams in which players played in each age category. METHODS: All players (N = 366) from an elite soccer academy of a Spanish club volunteered to participate in the study (U23-U10). RESULTS: There was a significant correlation between the RA of the players and the level of the team in which they played in each age category but no correlation between trimester of birth and level of the team. We found significant correlations between the players' physical capacities, anthropometry, RA and the level of the team in which they played for the same age category, mainly from U16 to U10. U23 did not show any correlation between RA and physical or anthropometric variables. CONCLUSION: Coaches should be cautious of choosing players based only on anthropometric or physical attributes.

Effects of different phenylcapsaicin doses on neuromuscular activity and mechanical performance in trained male subjects: a randomized, triple-blinded, crossover, placebo-controlled trial.

Objective: This study aimed to examine the effects of phenylcapsaicin (PC) supplementation on strength performance and neuromuscular activity in young trained male subjects. Materials and methods: A total of 25 trained subjects [full-squat (SQ) one repetition maximum (1RM) = 125.6 ± 21.0 kg] were enrolled in this randomized, triple-blinded, crossover, placebo-controlled trial. The subjects performed a first session and a post-24 h session for each condition. In the first session, the subjects ingested a high dose of PC (HD, 2.5 mg), a low dose (LD, 0.625 mg), or a placebo (PLA). Their performance in SQ was assessed under a 3% × 8 × 70% 1RM protocol in the first session. Their performances in countermovement jump (CMJ), SQ with 60% 1RM, and isometric squat were measured before and after the SQ protocol in both sessions. The neural activity of the vastus lateralis (VL) and vastus medialis (VM) was recorded via surface electromyography (EMG) and averaged in both sessions. Results: Significant differences between the conditions were reported for lifting velocity, velocity loss, and the 60% load in dynamic SQ (p range = 0.02-0.04). Electrical changes were not identified for any outcome, although neural activity changed across time (p range ≤0.001-0.006). A significant condition × time effect was observed in CMJ compared to PLA (p ≤0.001) and LD (p ≤0.001). Intra-set analyses revealed higher velocities in HD compared to those in LD (p = 0.01) and PLA (p range = 0.004-0.008). Conclusion: Therefore, PC may improve the strength performance and attenuate the mechanical fatigue induced by resistance training in SQ and CMJ exercises.

Impact of a Serious Game (#RedPingüiNO) to Reduce Facial Self-Touches and Prevent Exposure to Pathogens Transmitted via Hands: Quasi-Experimental Intervention.

BACKGROUND: After the COVID-19 pandemic, society has become more aware of the importance of some basic hygienic habits to avoid exposure to pathogens transmitted via hands. Given that a high frequency of touching mucous membranes can lead to a high risk of infection, it is essential to establish strategies to reduce this behavior as a preventive measure against contagion. This risk can be extrapolated to a multitude of health scenarios and transmission of many infectious diseases. #RedPingüiNO was designed as an intervention to prevent the transmission of SARS-CoV-2 and other pathogens through the reduction of facial self-touches by thoughtfully engaging participants in a serious game. OBJECTIVE: Facial self-touches should be understood as behaviors of limited control and awareness, used to regulate situations of cognitive and emotional demands, or as part of nonverbal communication. The objective of this study was to ensure that participants become aware of and reduce these behaviors through a game of self-perception. METHODS: The quasi-experimental intervention was applied to 103 healthy university students selected by convenience sampling and put into practice for 2 weeks, with 1 control group (n=24, 23.3%) and 2 experimental groups (experimental group with no additional social reinforcement interventions: n=36, 35%; experimental group with additional social reinforcement interventions: n=43, 41.7%). The objective was to improve knowledge and perception and reduce facial self-touches to prevent exposure to pathogens transmitted via hands not only in health multihazard scenarios but also in ordinary circumstances. The ad hoc instrument used to analyze the experience consisted of 43 items and was valid and reliable for the purpose of this study. The items were divided into 5 blocks extracted from the theoretical framework: sociological issues (1-5); hygiene habits (6-13); risk awareness (14-19); strategies for not touching the face (20-26); and questions after the intervention (27-42), designed as a postintervention tool assessing the game experience. Validation of the content was achieved through assessment by 12 expert referees. External validation was performed using a test-retest procedure, and reliability was verified using the Spearman correlation. RESULTS: The results of the ad hoc questionnaire, which were analyzed using the Wilcoxon signed-rank test and McNemar index to identify significant differences between test and retest for a 95% CI, showed that facial self-touches were reduced (item 20, P<.001; item 26, P=.04), and awareness of this spontaneous behavior and its triggers increased (item 15; P=.007). The results were reinforced by qualitative findings from the daily logs. CONCLUSIONS: The intervention exhibited a greater effect from sharing the game, with interactions between people; however, in both cases, it was helpful in reducing facial self-touches. In summary, this game is suitable for reducing facial self-touches, and owing to its free availability and design, it can be adapted to various contexts.

Effects of different phenylcapsaicin doses on resistance training performance, muscle damage, protein breakdown, metabolic response, ratings of perceived exertion, and recovery: a randomized, triple-blinded, placebo-controlled, crossover trial.

BACKGROUND: The aim of this study was to explore the effects of a low dose (LD) of 0.625 mg and a high dose (HD) of 2.5 mg of phenylcapsaicin (PC) on full squat (SQ) performance, active muscle (RPE-AM) and overall body (RPE-OB) ratings of perceived exertion, muscle damage, protein breakdown, metabolic response, and 24-h recovery in comparison to placebo (PLA). METHOD: Twenty-five resistance-trained males (age = 21.00 ± 2.15 years, SQ 1-repetition maximum [1RM] normalized = 1.66 ± 0.22 kg) were enrolled in this randomized, triple-blinded, placebo-controlled, crossover trial. Participants completed 2 weekly sessions per condition (LD, HD, and PLA). The first session consisted of pre-blood testing of lactate, urea, and aspartate aminotransferases (AST) and 2 SQ repetitions with 60% 1RM followed by the resistance exercise protocol, which consisted of SQ sets of 3 × 8 × 70% 1RM monitoring lifting velocity. RPE-OB and RPE-AM were assessed after each set. After the first session, 2 SQ repetitions with 60% 1RM were performed, and blood lactate and urea posttests were collected. After 24 h, AST posttest and 1 × 2 × 60% 1RM were determined as biochemical and mechanical fatigue outcomes. RESULTS: HD reported significant differences for RPE-AM, AST, and SQ performance compared to LD and PLA. Post-hoc analyses revealed that HD attained faster velocities in SQ than LD (p = 0.008). HD induced a lower RPE-AM when compared with LD (p = 0.02) and PLA (p = 0.004). PLA resulted in higher AST concentrations at 24-h post than HD (p = 0.02). No significant differences were observed for the rest of the comparisons. CONCLUSIONS: This study suggests that PC may favorably influence SQ performance, RPE-AM, and muscle damage compared to PLA. However, HD exhibited most of the biochemical and mechanical anti-fatigue effects instead of LD.

Hippocampal cholinergic receptors and the mTOR participation in fear-motivated inhibitory avoidance extinction memory.

Evidence has demonstrated the hippocampal cholinergic system and the mammalian target of rapamycin (mTOR) participation during the memory formation of aversive events. This study assessed the role of these systems in the hippocampus for the extinction memory process by submitting male Wistar rats to fear-motivated step-down inhibitory avoidance (IA). The post-extinction session administration of the nicotinic and muscarinic cholinergic receptor antagonists, mecamylamine and scopolamine, respectively, both at doses of 2 µg/µl/side, and rapamycin, an mTOR inhibitor (0.02 µg/µl/side), into the CA1 region of the dorsal hippocampus, impaired the IA extinction memory. Furthermore, the nicotinic and muscarinic cholinergic receptor agonists, nicotine and muscarine, respectively, had a dose-dependent effect on the IA extinction memory when administered intra-CA1, immediately after the extinction session. Nicotine (0.6 µg/µl/side) and muscarine (0.02 µg/µl/side), respectively, had no effect, while the higher doses (6 and 2 µg/µl/side, respectively) impaired the IA extinction memory. Interestingly, the co-administration of muscarine at the lower dose blocked the impairment that was induced by rapamycin. This effect was not observed when nicotine at the lower dose was co-administered. These results have demonstrated the participation of the cholinergic receptors and mTOR in the hippocampus for IA extinction, and that the cholinergic agonists had a dose-dependent effect on the IA extinction memory. This study provides insights related to the behavioural aspects and the neurobiological properties underlying the early stage of fear-motivated IA extinction memory consolidation and suggests that there is hippocampal muscarinic receptor participation independent of mTOR in this memory process.

Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity.

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity ex vivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Ex vivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Workloads of Different Soccer-Specific Drills in Professional Players.

Soccer is a predominantly tactical sport and, therefore, tactical training has become the most widely used strategy to improve players' performance. The objective of the present study was to assess the workload of soccer-specific drills in professional players over a two-season period in an established context. GPS technology was used to record the data. One hundred and thirty-two (n = 132) soccer-specific drills were studied and grouped by categories. The individual demands of each task were related to the individual competitive profile of each player. The level of physical demand was significantly different in relation to the specific soccer drills analysed. Total distance covered, high-speed running, and the total number of high accelerations and decelerations were significantly higher in competition than in drills used for training sessions (p < .001). The Large-Sided Games (LSG), Big-Position Games (BPG) and Position Games (PG) showed higher maximum running speed values than the rest of the exercises (p < .01). The sum of high accelerations and decelerations values was greater in the Small-Sided Games (SSG) than in BPG (p < .001), Small-Position Games (SPG) (p < .001) and Physical-Technical Circuits (PTC) (p < .001). Significant differences were observed in the exercises analysed according to the player's position. The current findings provide a detailed description of conditional demands placed on soccer players in different soccer-specific drills during training sessions, in a professional soccer context and according to their playing position, which may be helpful in the development of individualized training programs in other contexts.

Platelets and tyrosine kinase inhibitors: clinical features, mechanisms of action, and effects on physiology.

Tyrosine kinase inhibitors (TKIs) have emerged as a promising class of target-directed, small molecule inhibitors used to treat hematologic malignancies, inflammatory diseases, and autoimmune disorders. Recently, TKIs have also gained interest as potential antiplatelet-directed therapeutics that could be leveraged to reduce pathologic thrombus formation and atherothrombotic complications, while minimally affecting platelet hemostatic function. This review provides a mechanistic overview and summarizes the known effects of tyrosine kinase inhibitors on platelet signaling and function, detailing prominent platelet signaling pathways downstream of the glycoprotein VI (GPVI) receptor, integrin αIIbß3, and G protein-coupled receptors (GPCRs). This review focuses on mechanistic as well as clinically relevant and emerging TKIs targeting major families of tyrosine kinases including but not limited to Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (Syk), Src family kinases (SFKs), Janus kinases (JAK), and signal transducers and activators of transcription (STAT) and evaluates their effects on platelet aggregation and adhesion, granule secretion, receptor expression and activation, and protein phosphorylation events. In summation, this review highlights current advances and knowledge on the effects of select TKIs on platelet biology and furthers insight on signaling pathways that may represent novel druggable targets coupled to specific platelet functional responses.

Participation of Hippocampal 5-HT5A, 5-HT6 and 5-HT7 Serotonin Receptors on the Consolidation of Social Recognition Memory.

Social recognition is the ability of animals to identify and recognize a conspecific. The consolidation of social stimuli in long-term memory is crucial for the establishment and maintenance of social groups, reproduction and species survival. Despite its importance, little is known about the circuitry and molecular mechanisms involved in the social recognition memory (SRM). Serotonin (5-hydroxytryptamine, 5-HT) is acknowledged as a major neuromodulator, which plays a key role in learning and memory. Focusing on the more recently described 5-HT receptors, we investigated in the CA1 region of the dorsal hippocampus the participation of 5-HT5A, 5-HT6 and 5-HT7 receptors in the consolidation of SRM. Male Wistar rats cannulated in CA1 were subjected to a social discrimination task. In the sample phase the animals were exposed to a juvenile conspecific for 1 h. Immediately after, they received different pharmacological treatments. Twenty-four hours later, they were submitted to a 5 min retention test in the presence of the previously presented juvenile (familiar) and a novel juvenile. The animals that received infusions of 5-HT5A receptor antagonist SB-699551 (10 µg/µL), 5-HT6 receptor agonist WAY-208466 (0.63 µg/µL) or 5-HT7 receptor agonist AS-19 (5 µg/µL) intra-CA1 were unable to recognize the familiar juvenile. This effect was blocked by the coinfusion of WAY-208466 plus 5-HT6 receptor antagonist SB-271046 (10 µg/µL) or AS-19 plus 5-HT7 receptor antagonist SB-269970 (5 µg/µL). The present study helps to clarify the neurobiological functions of the 5-HT receptors more recently described and extends our knowledge about mechanisms underlying the SRM.

The Physiological Effects of Face Masks During Exercise Worn Due to COVID-19: A Systematic Review.

CONTEXT: Use of face masks in sport has been a particularly complex issue during the COVID-19 pandemic. OBJECTIVES: A systematic review to examine the physiological effects the different types of masks have on healthy adults when doing physical exercise. DATA SOURCES: PubMed, SPORTDiscus, Scopus, and Litcovid were searched up to March 20, 2021, following the PRISMA model. Articles published in the last 5 years with healthy adults. STUDY SELECTION: A total of 633 studies related to the use of masks during physical exercise were found, of which 8 articles met the criteria to be included. STUDY DESIGN: Systematic review. LEVEL OF EVIDENCE: Level 2. DATA EXTRACTION: The search process and the review of the articles were carried out by independent expert researchers. The risk of bias and the methodological quality of the different studies included in the systematic review were calculated following the Cochrane criteria using an adaptation for random cross-studies. Once the information was properly structured, the results were extracted, and the findings of the study analyzed. RESULTS: There were significant changes in the following physiological variables when engaging in physical exercise using masks: 25% in the heart rate and dyspnea, 37.5% in the rating of perceived exertion, 50% in the pulmonary variables, and 37.5% in discomfort. The oxygen saturation, blood pressure, systolic blood pressure, diastolic blood pressure, and the concentration of blood lactate did not present any significant effect in this study. CONCLUSION: The usage of masks by a healthy adult population during the performance of physical exercise has shown minimal effects with regard to physiological, cardiorespiratory, and perceived responses. Some symptoms can be dyspnea, effort perceived, or discomfort, among others. These findings indicate that the use of masks is not harmful to individuals' health. It does not present any significant detrimental effect on physical performance or risk to their well-being. However, further experiments are required to corroborate the findings of this review.

Involvement of medial prefrontal cortex canonical Wnt/ß-catenin and non-canonical Wnt/Ca2+ signaling pathways in contextual fear memory in male rats.

Wnt proteins activate different signaling pathways, such as the canonical Wnt/ß-catenin signaling pathway and non-canonical ß-catenin-independent signaling pathway and have been related to several functions in central nervous system, including learning and memory. However, whether these signaling pathways are required in the medial prefrontal cortex (mPFC) for fear memory acquisition, consolidation and retrieval remains unclear. To address this question, we submitted male rats to a contextual fear conditioning (CFC) paradigm, and administered canonical Wnt/ß-catenin and non-canonical Wnt/Ca2+ signaling pathways inhibitors, DKK1 and SFRP1, respectively, into the prelimbic (PrL) subdivision of the mPFC at different moments and evaluated short-term and long-term memory acquisition, consolidation and retrieval. We found that blocking canonical Wnt/ß-catenin and non-canonical Wnt/Ca2+ signaling pathways 15 min before or immediately after CFC training had no effect on STM and LTM of CFC, while their blockade 15 min before the retention test prevented the retrieval of STM and LTM of CFC. These results highlight the importance of the mPFC in fear memory retrieval demonstrating that both canonical Wnt/ß-catenin and non-canonical Wnt/Ca2+ signaling pathways participate in this process. To understand how brain systems act on fear memories could provide a new target for the treatment of fear related disorders such as post-traumatic stress disorder and other anxiety disorders.

Modulation of Carbonic Anhydrases Activity in the Hippocampus or Prefrontal Cortex Differentially Affects Social Recognition Memory in Rats.

Growing evidence indicates that brain carbonic anhydrases (CAs) are key modulators in cognition, particularly in recognition and aversive memories. Here we described a role for these enzymes also in social recognition memory (SRM), defined as the ability to identify and recognize a conspecific, a process that is of paramount importance in gregarious species, such as rodents and humans. Male adult Wistar rats were submitted to a social discrimination task and, immediately after the sample phase, received bilateral infusions of vehicle, the CAs activator D-phenylalanine (D-Phen, 50 nmols/side), the CAs inhibitor acetazolamide (ACTZ; 10 nmols/side) or the combination of D-Phen and ACTZ directly in the CA1 region of the dorsal hippocampus or in the medial prefrontal cortex (mPFC). Animals were tested 30 min (short-term memory) or 24 h later (long-term memory). We found that inhibition of CAs with infusion of ACTZ either in the CA1 or in the mPFC impaired short-term SRM and that this effect was completely abolished by the combined infusion of D-Phen and ACTZ. We also found that activation of CAs with D-Phen facilitated the consolidation of long-term SRM in the mPFC but not in CA1. Finally, we show that activation of CAs in CA1 and in the mPFC enhances the persistence of SRM for up to 7 days. In both cases, the co-infusion of ACTZ fully prevented D-Phen-induced procognitive effects. These results suggest that CAs are key modulators of SRM and unveil a differential involvement of these enzymes in the mPFC and CA1 on memory consolidation.

Chronic edible dosing of Δ9-tetrahydrocannabinol (THC) in nonhuman primates reduces systemic platelet activity and function.

Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.

PKMζ Maintains Remote Contextual Fear Memory by Inhibiting GluA2-Dependent AMPA Receptor Endocytosis in the Prelimbic Cortex.

Fear memories allow animals to recognize and adequately respond to dangerous situations. The prelimbic cortex (PrL) is a crucial node in the circuitry that encodes contextual fear memory, and its activity is central for fear memory expression over time. However, while PrL has been implicated in contextual fear memory storage, the molecular mechanisms underlying its maintenance remain unclear. Protein kinase M zeta (PKMζ) is a persistently active enzyme which has been shown to maintain many forms of memories by inhibiting the endocytosis of GluA2-containing AMPA receptors. Therefore, we hypothesized that PKMζ action upon GluA2-containing AMPARs could be a mechanism for contextual fear memory maintenance in the PrL. To test this hypothesis, we trained rats in a contextual fear conditioning (CFC) paradigm and administered intra-PrL infusions of the PKMζ inhibitor ZIP, the GluA2-dependent endocytosis inhibitor GluA23Y or the inactive peptide GluA23Y(s), either two or twenty days after conditioning, and assessed long-term memory retention twenty-four hours later. We found that acute inhibition of GluA2-dependent AMPAR endocytosis in the PrL does not affect recent or remote contextual fear memory maintenance. Also, PKMζ inhibition in the PrL does not impair the maintenance of recent contextual fear memory. However, we found that inhibition of prelimbic PKMζ at a remote time point disrupts contextual fear memory maintenance, and that blocking GluA2-dependent removal of AMPARs prevents this impairment. Our results confirm the central role of PrL in fear memory and identify PKMζ-induced inhibition of GluA2-containing AMPAR endocytosis as a key mechanism governing remote contextual fear memory maintenance.

Structure of the training program during the COVID-19 confinement in spanish professional football, a coach survey.

OBJECTIVES: To describe and analyze the training programs carried out during the COVID-19 forced confinement in men´s professional football in Spain. METHODS: Observational Study based on a telematic ad-hoc questionnaire developed to meet the objective of the study. The sample consisted of thirty-six coaches representing thirty-six professional men's soccer teams in the Spanish first and second division. RESULTS: Training programs developed during confinement prioritized conditioning and functional emphasis, in addition to general and nonspecific resistance training, due to contextual limitations. CONCLUSION: The structure of training during the COVID-19 confinement was limited by contextual circumstances. This study has made possible to record the training and strategies used in professional football during a confinement due to a worldwide state of alarm, with the aim of resuming competitive activity in the best possible conditions.

Janus kinase inhibitors ruxolitinib and baricitinib impair glycoprotein-VI mediated platelet function.

Several Janus kinase (JAK) inhibitors (jakinibs) have recently been approved to treat inflammatory, autoimmune and hematological conditions. Despite emerging roles for JAKs and downstream signal transducer and activator of transcription (STAT) proteins in platelets, it remains unknown whether jakinibs affect platelet function. Here, we profile platelet biochemical and physiological responses in vitro in the presence of five different clinically relevant jakinibs, including ruxolitinib, upadacitinib, oclacitinib, baricitinib and tofacitinib. Flow cytometry, microscopy and other assays found that potent JAK1/2 inhibitors baricitinib and ruxolitinib reduced platelet adhesion to collagen, as well as platelet aggregation, secretion and integrin αIIbß3 activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP-XL). Western blot analysis demonstrated that jakinibs reduced Akt phosphorylation and activation following GPVI activation, where ruxolitinib and baricitinib prevented DAPP1 phosphorylation. In contrast, jakinibs had no effects on platelet responses to thrombin. Inhibitors of GPVI and JAK signaling also abrogated platelet STAT5 phosphorylation following CRP-XL stimulation. Additional pharmacologic experiments supported roles for STAT5 in platelet secretion, integrin activation and cytoskeletal responses. Together, our results demonstrate that ruxolitinib and baricitinib have inhibitory effects on platelet function in vitro and support roles for JAK/STAT5 pathways in GPVI/ITAM mediated platelet function.

Interferons Are Pro-Inflammatory Cytokines in Sheared-Stressed Human Aortic Valve Endothelial Cells.

Calcific aortic valve disease (CAVD) is an athero-inflammatory process. Growing evidence supports the inflammation-driven calcification model, mediated by cytokines such as interferons (IFNs) and tumor necrosis factor (TNF)-α. Our goal was investigating IFNs' effects in human aortic valve endothelial cells (VEC) and the potential differences between aortic (aVEC) and ventricular (vVEC) side cells. The endothelial phenotype was analyzed by Western blot, qPCR, ELISA, monocyte adhesion, and migration assays. In mixed VEC populations, IFNs promoted the activation of signal transducers and activators of transcription-1 and nuclear factor-κB, and the subsequent up-regulation of pro-inflammatory molecules. Side-specific VEC were activated with IFN-γ and TNF-α in an orbital shaker flow system. TNF-α, but not IFN-γ, induced hypoxia-inducible factor (HIF)-1α stabilization or endothelial nitric oxide synthase downregulation. Additionally, IFN-γ inhibited TNF-α-induced migration of aVEC. Also, IFN-γ triggered cytokine secretion and adhesion molecule expression in aVEC and vVEC. Finally, aVEC were more prone to cytokine-mediated monocyte adhesion under multiaxial flow conditions as compared with uniaxial flow. In conclusion, IFNs promote inflammation and reduce TNF-α-mediated migration in human VEC. Moreover, monocyte adhesion was higher in inflamed aVEC sheared under multiaxial flow, which may be relevant to understanding the initial stages of CAVD.

The Toll-Like Receptor 2 Ligand Pam2CSK4 Activates Platelet Nuclear Factor-κB and Bruton's Tyrosine Kinase Signaling to Promote Platelet-Endothelial Cell Interactions.

Circulating platelets establish a variety of immunological programs and orchestrate inflammatory responses at the endothelium. Platelets express the innate immunity family of Toll-like receptors (TLRs). While TLR2/TLR1 ligands are known to activate platelets, the effects of TLR2/TLR6 ligands on platelet function remain unclear. Here, we aim to determine whether the TLR2/TLR6 agonists Pam2CSK4 and FSL-1 activate human platelets. In addition, human umbilical vein endothelial cells (HUVECs) and platelets were co-cultured to analyze the role of platelet TLR2/TLR6 on inflammation and adhesion to endothelial cells. Pam2CSK4, but not FSL-1, induced platelet granule secretion and integrin αIIbß3 activation in a concentration-dependent manner. Moreover, Pam2CSK4 promoted platelet aggregation and increased platelet adhesion to collagen-coated surfaces. Mechanistic studies with blocking antibodies and pharmacologic inhibitors demonstrated that the TLR2/Nuclear factor-κB axis, Bruton's-tyrosine kinase, and a secondary ADP feedback loop are involved in Pam2CSK4-induced platelet functional responses. Interestingly, Pam2CSK4 showed cooperation with immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling to enhance platelet activation. Finally, the presence of platelets increased inflammatory responses in HUVECs treated with Pam2CSK4, and platelets challenged with Pam2CSK4 showed increased adhesion to HUVECs under static and physiologically relevant flow conditions. Herein, we define a functional role for platelet TLR2-mediated signaling, which may represent a druggable target to dampen excessive platelet activation in thrombo-inflammatory diseases.