P2Y1 receptor activation elicits its partition out of membrane rafts and its rapid internalization from human blood vessels: implications for receptor signaling

The nucleotide P2Y(1) receptor (P2Y(1)R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y(1)R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y(1)R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1)R and obliterated the P2Y(1)R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y(1)R agonist, not only displaced within 4 min the P2Y(1)R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N(6)-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1)R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1)R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y(1)R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1)R to membrane rafts, highlighting the role of this microdomain in P2Y(1)R signaling.     

Coherent delta-band oscillations between cortical areas correlate with decision making

Coherent oscillations in the theta-to-gamma frequency range have been proposed as a mechanism that coordinates neural activity in large-scale cortical networks in sensory, motor, and cognitive tasks. Whether this mechanism also involves coherent oscillations at delta frequencies (1–4 Hz) is not known. Rather, delta oscillations have been associated with slow-wave sleep. Here, we show coherent oscillations in the delta frequency band between parietal and frontal cortices during the decision-making component of a somatosensory discrimination task. Importantly, the magnitude of this delta-band coherence is modulated by the different decision alternatives. Furthermore, during control conditions not requiring decision making, delta-band coherences are typically much reduced. Our work indicates an important role for synchronous activity in the delta frequency band when large-scale, distant cortical networks coordinate their neural activity during decision making.Studies of the neural correlates of decision making in behaving monkeys have mainly been based on the analysis of firing rate patterns of neurons in individual cortical circuits, recorded one by one in succession, while trained monkeys perform sensory, motor, and cognitive tasks (1–3). These studies showed that the neuronal activities distributed across parietal and frontal lobe cortices correlate with processes that lead to decision making (4–7). However, how these spatially distant, cortical circuits coordinate their activities into a unified functional network during decision making remains poorly understood.It has been proposed that coherent oscillations of neuronal activities constitute a putative dynamical mechanism for mediating the interaction between different subsets of brain areas (8–11). Simultaneous recording from multiple intracortical areas in monkeys showed that the coherent higher frequency (beta and gamma bands) oscillations are linked to a broad variety of cognitive functions (12–18). Cortical oscillations at lower frequencies (theta and alpha bands) have also been discussed in terms of long-range integrative processes (19, 20). In fact, recent evidence showed theta-band coupling between visual area V4 and prefrontal cortex during short-term memory (21) and among the rat prefrontal cortex, ventral tegmental area, and hippocampus during working memory (22). It remains, however, probing whether coherent delta-band oscillations play a functional role in the interaction between cortical circuits. Delta-band oscillations are typically associated with slow-wave sleep (SWS; ref. 23), but an important question is whether delta-band oscillations during SWS and waking states represent the same underlying phenomenon (24). Recent findings associated delta-band oscillations in individual cortical areas with attention (25). In monkey primary visual cortex (26) and human motor cortex (27), delta-band oscillations entrain to the rhythm of external sensory events in an attention-dependent manner.Here, we examined whether coherent oscillations coordinate the activity of five simultaneously recorded cortical areas in the monkey performing a somatosensory discrimination task (7). We specifically focused on whether coherent delta-band oscillations play a significant functional role in linking cortical circuits during decision making.     

The escape room as evaluation method: A qualitative study of nursing students’ experiences

Abstract

Purpose: Gamification or learning using game elements is a process that seeks to capture students’ interest. One of the most popular games in recent years is the Escape Room, but the study of its use in education and with health professionals is still limited. The aim of this study was to find out the perceptions and experiences of final year nursing students in an Objective Structured Clinical Examination (OSCE) by means of an Escape Room.

Methods: In January 2019, 9 focus groups (FG) were held, with a total of 95 final year nursing students. We applied a qualitative content analysis approach and ATLAS.ti version 8 was used for data analysis.

Results: The data revealed 3 main themes and 8 sub-themes. The three main themes, which were mapped to the conceptual framework, were student learning outcomes, emotional impact on students and conclusions on the serious games experience. Both the main themes and the sub-themes were illustrated using representative quotes from the participants.

Conclusions: These results can help to apply these methodologies, such as the Escape Room, alongside other pre-existing ones, complementing the way in which students are assessed and the development of important nursing skills, such as teamwork and communication.     

Analysis of the Ectomycorrhizal Enzymatic Functional Diversity of <Emphasis Type="Italic">Fagus mexicana</Emphasis>

In forest ecosystems, ectomycorrhizal symbiosis is able to produce a large range of extracellular and cell wall-bound enzymes which hydrolyze compounds contained in soil organic matter. Enzymatic activity profiles of individual ectomycorrhiza have value of functional traits to characterize the diversity of ectomycorrhizal (ECM) communities. Fagus mexicana is an endemic and only species that has restricted distribution to cloud forest of Sierra Madre Oriental in México and there are few works focused to the relationship between this forest species and ectomycorrhizal, which enhance its growth and development. This work analyzes the enzyme functional diversity of ectomycorrhizal fungi associated to F. mexicana trees of different ages. In this study, the ECM species: Cantharellus cibarius Fr., Laccaria amethystina Cook, Lactarius subdulcis (Pers.) gray and Xathoconium separans (Peck) Halling & Both, were founded near these trees and the influence of the niche on the mean catabolic activity of ectomycorrhizal community, varied according to the dominant substrate; where L. amethystina and X. separans presented the highest β-glucosidase and acid phosphatase activities in F. mexicana tips. The enzymatic profiles demonstrated a functional plasticity of the ectomycorrhizal species associated to the age of trees tested and also could be a trait of their capability of adaptation to the cloud forest ecosystem.     

Regulation of the Cardiac Sodium/Bicarbonate Cotransporter by Angiotensin II: Potential Contribution to Structural,Ionic and Electrophysiological Myocardial Remodelling

The sodium/ bicarbonate cotransporter (NBC) is, with the Na⁺/H⁺ exchanger (NHE), an important alkalinizing mechanism that maintains cellular intracellular pH (pHi). In the heart exists at least three isoforms of NBC, one that promotes the co-influx of 1 molecule of Na⁺ per 1molecule of HCO₃^-(electroneutral isoform; nNBC) and two others that generates the co-influx of 1 molecule of Na⁺ per 2 molecules of HCO₃^- (electrogenic isoforms; eNBC). In addition, the eNBC generates an anionic repolarizing current that modulate the cardiac action potential (CAP), adding to such isoforms the relevance to modulate the electrophysiological function of the heart. Angiotensin II (Ang II) is one of the main hormones that regulate cardiac physiology. The alkalinizing mechanisms (NHE and NBC) are stimulated by Ang II, increasing pHi and intracellular Na⁺ concentration, which indirectly, due to the stimulation of the Na⁺/Ca²⁺ exchanger (NCX) operating in the reverse form, leads to an increase in the intracellular Ca²⁺ concentration. Interestingly, it has been shown that Ang II exhibits an opposite effect on NBC isoforms: it activates the nNBC and inhibits the eNBC. This inhibition generates a CAP prolongation, which could directly increase the intracellular Ca²⁺ concentration. The regulation of the intracellular Na⁺ and Ca²⁺ concentrations is crucial for the cardiac cellular physiology, but these ions are also involved in the development of cardiac hypertrophy and the damage produced by ischemia-reperfusion, suggesting a potential role of NBC in cardiac diseases.     

Type 2 Diabetes Mellitus in Patients with Mild Obesity: Preliminary Results of Surgical Treatment

In the last years, type 2 diabetes mellitus (T2DM) and obesity have become a serious public health problem, behaving as epidemic diseases. There is great interest in exploring different options for the treatment of T2DM in nonmorbidly obese patients. The purpose of this study is to report parameters of glycemic control in patients with T2DM and mild obesity who underwent laparoscopic Roux-en-Y gastric bypass (RYGBP). This prospective clinical trial includes patients with T2DM with a body mass index (BMI) between 30 and 35 kg/m² who underwent laparoscopic RYGBP from July 2008 through October 2010. Thirty-one patients were included in the study, 15 men and 16 women, with an average age of 48.7?±?8.6 years. The average time since onset of T2DM was 5.8 years. The average postoperative follow-up was 30.4 months. The average preoperative blood glucose and glycosylated hemoglobin were 152?±?70 mg/dl and 7.7?±?2.1 %, respectively. All of them were using oral hypoglycemic agents, and four patients were insulin dependent. Only one patient had a postoperative complication (hemoperitoneum). At 36 months follow-up, the average BMI decreased to 24.7 kg/m², all patients (31) showed improvement in their glycemic control, and 29 of them (93.6 %) met the criteria for remission of T2DM in the last control. Laparoscopic RYGBP is a safe and effective procedure that improves glycemic control in patients with T2DM and mild obesity at midterm follow-up.     

Shiga Toxin 1-Induced Inflammatory Response in Lipopolysaccharide-Sensitized Astrocytes Is Mediated by Endogenous Tumor Necrosis Factor Alpha

Hemolytic-uremic syndrome (HUS) is generally caused by Shiga toxin (Stx)-producing Escherichia coli. Endothelial dysfunction mediated by Stx is a central aspect in HUS development. However, inflammatory mediators such as bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) contribute to HUS pathophysiology by potentiating Stx effects. Acute renal failure is the main feature of HUS, but in severe cases, patients can develop neurological complications, which are usually associated with death. Although the mechanisms of neurological damage remain uncertain, alterations of the blood-brain barrier associated with brain endothelial injury is clear. Astrocytes (ASTs) are the most abundant inflammatory cells of the brain that modulate the normal function of brain endothelium and neurons. The aim of this study was to evaluate the effects of Stx type 1 (Stx1) alone or in combination with LPS in ASTs. Although Stx1 induced a weak inflammatory response, pretreatment with LPS sensitized ASTs to Stx1-mediated effects. Moreover, LPS increased the level of expression of the Stx receptor and its internalization. An early inflammatory response, characterized by the release of tumor necrosis factor alpha (TNF-α) and nitric oxide and PMN-chemoattractant activity, was induced by Stx1 in LPS-sensitized ASTs, whereas activation, evidenced by higher levels of glial fibrillary acid protein and cell death, was induced later. Furthermore, increased adhesion and PMN-mediated cytotoxicity were observed after Stx1 treatment in LPS-sensitized ASTs. These effects were dependent on NF-κB activation or AST-derived TNF-α. Our results suggest that TNF-α is a pivotal effector molecule that amplifies Stx1 effects on LPS-sensitized ASTs, contributing to brain inflammation and leading to endothelial and neuronal injury.The epidemic form of hemolytic-uremic syndrome (HUS) has been associated with enterohemorrhagic infections caused by Shiga toxin (Stx)-producing Escherichia coli (STEC) organisms (33). HUS is the most common cause of acute renal failure in children and is related to the endothelial damage of glomeruli and/or arterioles of the kidney and epithelial cell damage induced by Stx through the interaction with its globotriaosylceramide (Gb₃) receptor (35). Although Stx is the main pathogenic factor and is necessary for epidemic HUS development, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity. In fact, both bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) play a key role in the full development of HUS (15). Moreover, PMN leukocytosis in patients correlates with a poor prognosis (17).Endothelial cell damage is not limited to the kidney but extends to other organs; in severe cases, the brain can be affected. In fact, central nervous system (CNS) complications indicate severe HUS, and brain damage involvement is the most common cause of death (14).However, the pathogenesis of CNS impairment is not yet fully understood. Although it has been demonstrated that human brain endothelial cells (BECs) are relatively resistant to Stx, inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), markedly increase human BEC sensitivity to Stx cytotoxicity (11).BECs are part of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances and leukocytes present in the bloodstream. Thus, the integrity of BBB function is theorized to be a key component in CNS-associated pathologies, and BEC damage is thought to be one of the possible mechanisms involved in the disruption of the BBB in HUS. In fact, LPS from bacterial infections leads to the release of TNF-α, interleukin-1β (IL-1β), and reactive oxygen species (ROS), all of which have the ability to open the BBB.Several in vivo studies demonstrated previously that Stx is able to impair BBB function, increasing its permeability (21). Moreover, Stx itself is able to cross the endothelial barrier and enter into the CNS, since Stx activity in cerebrospinal fluid was previously observed (19, 23), and Stx was previously immunodetected in many brain cells including astrocytes (ASTs) and neurons (44).ASTs, which are inflammatory cells found throughout the CNS, are in close contact with BECs by end-foot processes (24), and their interaction with the cerebral endothelium determines BBB function (2, 4). In addition, ASTs interact with neurons through gap junctions and release neurotrophins that are essential for neuronal survival (6). However, in response to brain injury, ASTs become activated and release inflammatory mediators such as nitric oxide (NO) and TNF-α, altering the permeability of the BBB and affecting neuronal survival and tissue integrity (1, 9). In addition, AST-derived cytokines and chemokines can stimulate the peripheral immune system and attract peripheral inflammatory leukocytes to the site of injury (46).ASTs are therefore in a critical position to influence neuronal viability and BEC integrity once Stx and factors associated with the STEC infection reach the brain parenchyma. We hypothesize that the effects of LPS and Stx on ASTs may be involved in the brain damage observed with severe cases of HUS. Thus, the aim of this study was to evaluate whether Stx type 1 (Stx1) alone or in combination with LPS is capable of inducing an inflammatory response in ASTs.     

Early nerve ending rescue from oxidative damage and energy failure by <Emphasis Type="SmallCaps">l</Emphasis>-carnitine as post-treatment in two neurotoxic models in rat: recovery of antioxidant and reductive capacities

Cell rescue is a primary need during acute and chronic insults to the central nervous system. Functional preservation during the early stages of toxicity in a given degenerative event may represent a significant amelioration of detrimental processes linked to neuronal cell loss. Excitotoxicity and depleted cellular energy are toxic events leading to cell death in several neurodegenerative disorders. In this work, the effects of the well-known antioxidant and energy precursor, l-carnitine (l-CAR), were tested as a post-treatment in two neurotoxic models under in vitro and in vivo conditions. The experimental models tested included: (1) a typical excitotoxic and pro-oxidant inducer, quinolinic acid (QUIN); and (2) a mitochondrial energy inhibitor, 3-nitropropionic acid (3-NP). For in vitro studies, increasing concentrations of l-CAR (10–1,000 μM) were added to the isolated brain synaptosomes at different times (1, 3 and 6 h) after the incubation with toxins (100 μM QUIN and 1 mM 3-NP), and 30 min later, lipid peroxidation (LP) and mitochondrial dysfunction (MD) were evaluated. For in vivo purposes, l-CAR (100 mg/kg, i.p.) was given to rats either as a single administration 120 min after the intrastriatal infusion of QUIN (240 nmol/μl) or 3-NP (500 nmol/μl), or for 7 consecutive days (starting 120 min post-lesion). LP and MD were evaluated 4 h and 7 days post-lesions in isolated striatal synaptosomes. Our results show that, despite some variations depending on the toxic model tested, the time of exposure, or the biomarker evaluated, nerve ending protection can be mostly achieved by l-CAR within the first hours after the toxic insults started, suggesting that targeting the ongoing oxidative damage and/or energy depletion during the first stages of neurotoxic events is essential to rescue nerve endings. D. Elinos-Calderón and Y. Robledo-Arratia equally contributed to this work.