Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

Untargeted metabolomics analysis on kidney tissues from mice reveals potential hypoxia biomarkers.

Chronic hypoxia may have a huge impact on the cardiovascular and renal systems. Advancements in microscopy, metabolomics, and bioinformatics provide opportunities to identify new biomarkers. In this study, we aimed at elucidating the metabolic alterations in kidney tissues induced by chronic hypoxia using untargeted metabolomic analyses. Reverse phase ultrahigh performance liquid chromatography-mass spectroscopy/mass spectroscopy (RP-UPLC-MS/MS) and hydrophilic interaction liquid chromatography (HILIC)-UPLC-MS/MS methods with positive and negative ion mode electrospray ionization were used for metabolic profiling. The metabolomic profiling revealed an increase in metabolites related to carnitine synthesis and purine metabolism. Additionally, there was a notable increase in bilirubin. Heme, N-acetyl-L-aspartic acid, thyroxine, and 3-beta-Hydroxy-5-cholestenoate were found to be significantly downregulated. 3-beta-Hydroxy-5-cholestenoate was downregulated more significantly in male than female kidneys. Trichome Staining also showed remarkable kidney fibrosis in mice subjected to chronic hypoxia. Our study offers potential intracellular metabolite signatures for hypoxic kidneys.

External and internal load during small-sided games in soccer: use or not floaters.

BACKGROUND: The purpose of the present study was to analyze the internal and external loads on regular and floater players during standardized small-sided games (SSGs) with different numbers of players (teams of 3, 5, or 7 players). METHODS: Fifteen male semi-professional football players played different SSGs maintaining the same relative area per player. Total distance (TD), distance covered at different speeds (DC), the number of accelerations and decelerations, maximal (HRmax) and mean (HRmean) heart rate and rate of perceived exertion (RPE) were registered. RESULTS: Regular players showed greater internal and external loads in SSGs with 3 and 5 players without floaters than with floaters (ES 0.60-to-1.27). Likewise, with floaters, regular players in the SSGs with 3 performed more accelerations (ES 1.40 and 1.17) and with 7 achieved higher TD, DC>14 km·h-1, HRmax and HRmean (ES 0.66-to-2.79) than any other. During SSGs with 7 players the floaters showed a higher TD and decelerations than in other SSGs (ES 0.47-to-1.70), and a higher DC (0-6.9 km·h-1,14-17.9 km·h-1) and RPE than in SSGs with 3 players (ES 0.59-to-0.89). During SSGs with 5, the floaters showed a higher TD, HRmax, HRmean and RPE than in SSGs with 3 (ES 0.86-to-1.45). In all SSGs, regular players showed higher TD, DC (14-17.9 km·h-1), accelerations, decelerations and HRmean than floaters (ES 1.24-to-6.23). CONCLUSIONS: Coaches must carefully design SSGs because the number of players and the presence or absence of floaters can affect the external-internal load expressed.

Predicting Loading Intensity Measuring Velocity in Barbell Hip Thrust Exercise.

ABSTRACT: de Hoyo, M, Núñez, FJ, Sañudo, B, Gonzalo-Skok, O, Muñoz-López, A, Romero-Boza, S, Otero-Esquina, C, Sánchez, H, and Nimphius, S. Predicting loading intensity measuring velocity in barbell hip thrust exercise. J Strength Cond Res 35(8): 2075-2081, 2021-The barbell hip thrust is an increasingly used exercise to target the hip extensors. Direct and indirect measurement of 1 repetition maximum (1RM) to determine the relative load of each exercise is time-consuming; therefore, practitioners may be more in favor of monitoring velocity and determining relative load through velocity-based prediction models for an exercise. This study aimed to assess the relationship between mean velocity (MV) and mean propulsive velocity (MPV) at different relative training loads (%1RM) in the barbell hip thrust exercise. One hundred two male sport science students performed an incremental 1RM testing protocol for the barbell hip thrust exercise, and a linear position transducer measured MV and MPV of the barbell. The 1RM was reached at 0.25 ± 0.03 m·s-1, and the regression model generated to estimate a relative load showed an acceptable standard error of estimate (7.01 ± 1.05% 1RM and 7.36 ± 1.05% 1RM for MV and MPV, respectively) with a very large explained variance (R2 = 0.94). These results may have important practical applications for the prescription and monitoring of the accessory exercise of the hip thrust for monitoring training load and predicting 1RM without undertaking a RM test.

Phosphodiesterase isoforms and cAMP compartments in the development of new therapies for obstructive pulmonary diseases.

The second messenger molecule 3'5'-cyclic adenosine monophosphate (cAMP) imparts several beneficial effects in lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). While cAMP is bronchodilatory in asthma and COPD, it also displays anti-fibrotic properties that limit fibrosis. Phosphodiesterases (PDEs) metabolize cAMP and thus regulate cAMP signaling. While some existing therapies inhibit PDEs, there are only broad family specific inhibitors. The understanding of cAMP signaling compartments, some centered around lipid rafts/caveolae, has led to interest in defining how specific PDE isoforms maintain these signaling microdomains. The possible altered expression of PDEs, and thus abnormal cAMP signaling, in obstructive lung diseases has been poorly explored. We propose that inhibition of specific PDE isoforms can improve therapy of obstructive lung diseases by amplifying specific cAMP signals in discreet microdomains.

Glucocorticoids rapidly activate cAMP production via Gαs to initiate non-genomic signaling that contributes to one-third of their canonical genomic effects.

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non-genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3',5'-cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real-time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane- impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid-stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein-coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non-genomic actions of budesonide were analyzed by RNA-Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs . A 140-gene budesonide signature was identified, of which 48 genes represent a non-genomic signature that requires Gαs signaling. Collectively, this non-genomic cAMP signaling modality contributes to one-third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.

Agonist-specific desensitization of PGE2-stimulated cAMP signaling due to upregulated phosphodiesterase expression in human lung fibroblasts.

Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3',5'-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.

Transforming Growth Factor-ß1 Decreases ß2-Agonist-induced Relaxation in Human Airway Smooth Muscle.

Helper T effector cytokines implicated in asthma modulate the contractility of human airway smooth muscle (HASM) cells. We have reported recently that a profibrotic cytokine, transforming growth factor (TGF)-ß1, induces HASM cell shortening and airway hyperresponsiveness. Here, we assessed whether TGF-ß1 affects the ability of HASM cells to relax in response to ß2-agonists, a mainstay treatment for airway hyperresponsiveness in asthma. Overnight TGF-ß1 treatment significantly impaired isoproterenol (ISO)-induced relaxation of carbachol-stimulated, isolated HASM cells. This single-cell mechanical hyporesponsiveness to ISO was corroborated by sustained increases in myosin light chain phosphorylation. In TGF-ß1-treated HASM cells, ISO evoked markedly lower levels of intracellular cAMP. These attenuated cAMP levels were, in turn, restored with pharmacological and siRNA inhibition of phosphodiesterase 4 and Smad3, respectively. Most strikingly, TGF-ß1 selectively induced phosphodiesterase 4D gene expression in HASM cells in a Smad2/3-dependent manner. Together, these data suggest that TGF-ß1 decreases HASM cell ß2-agonist relaxation responses by modulating intracellular cAMP levels via a Smad2/3-dependent mechanism. Our findings further define the mechanisms underlying ß2-agonist hyporesponsiveness in asthma, and suggest TGF-ß1 as a potential therapeutic target to decrease asthma exacerbations in severe and treatment-resistant asthma.

Analysis of the acceleration profile according to initial speed and positional role in elite professional male soccer players.

BACKGROUND: The aim of the current study was to analyze the acceleration profile in elite professional soccer players according to their initial speed but also considering players' position. METHODS: Players' accelerations profiles were analyzed using a relative acceleration profile according to the initial speed (S1, from 0 to 7 km/h; S2, from 7.1 to 14.3 km/h; and S3, ≥14.4 km/h) and the maximum acceleration. RESULTS: Within-group analyzes showed that center backs (CB) performed more high intensity accelerations (likely) when they started in S1 than S2 (effect size [ES] 0.50). Strikers (S) and wide midfielders (W-MD) achieved more accelerations (likely to almost certain) starting in S3 than S1 (ES=0.80 and ES=0.59, respectively) and S2 (ES=0.67 and ES=1.09, respectively). Full backs (FB) completed more accelerations (almost certain) starting in S1 and S3 than S2 (ES=1.39 and ES=1.36, respectively). Finally, midfielders (MD) executed a greater number of high intensity accelerations (likely to almost certain) when they started in S1 than S2 (ES=0.83) and S3 (ES=0.66), and in S3 than S2 (ES=4.72). Between-group analyzes showed that S, W-MD, and FB performed a greater total number of high intensity accelerations (very likely to almost certain) than CB (ES=1.94, ES=1.57, and ES=1.51, respectively) and MD (ES=1.23, ES=0.92; and ES=0.81, respectively). Furthermore, MD performed substantially greater total number of high intensity accelerations (likely) than CB (ES=0.56). CONCLUSIONS: Results suggest that CB achieved more high-intensity accelerations starting in low and moderate speed, S and W-MD in high speed, and FB combined low and high speed.

Relationship of the drying process of a corn grain with the thermal emissivity and optical interference in the mid infrared range.

A study of the effect of the thin layer of free water in corn kernels on the emissivity and interference in the mid infrared range was performed. The emissivity was measured through thermal infrared images by direct method for 8 days, allowing observance that the thickness of free water modifies the quantity of emitted energy and emissivity; however, in the first days when the layer of free water is not optically thick the interference caused by the thin film of superficial water averts a correct measurement of the emissivity. This interference effect was studied and characterized, finding that the number of oscillations in the energy of the grain, observed and counted in a very small area, can be used to compute the thickness of the free water layer contained between the endosperm and the pericarp of the grain.

Intratissue percutaneous electolysis combined with active physical therapy for the treatment of adductor longus enthesopathy-related groin pain: a randomized trial.

BACKGROUND: Adductor longus enthesopathy-related groin pain (ALErGP) is the most common cause of groin pain in soccer players. The aim of this study was to evaluate the therapeutic utility of intratissue percutaneous electrolysis (EPI®) technique in combination with an active physical therapy (APT) program to treat ALErGP. METHODS: Twenty-four non-professional male soccer players diagnosed with ALErGP were included in this study and randomly divided into two groups. Group A was treated with EPI® technique in combination with a standardized APT program. Group B only underwent the APT program. The Numeric Rating Scale (NRS) and the Patient Specific Functional Scale (PSFS) were used to assess the effectiveness of the two interventions. The follow-up covered a 6-month period. RESULTS: Both groups significantly improved pain and functional scores after treatment and maintained this therapeutic result throughout the follow-up. The combined intervention of APT program and EPI® ensured a greater and faster reduction of pain in group A. In addition, functional recovery tended to be greater in group A than B after the treatment and throughout the follow-up by 7.8±3.8% (P=0.093). CONCLUSIONS: EPI® treatment in association with APT ensured a greater and more rapid reduction of pain and tended to promote greater functional recovery in soccer players with ALErGP compared to APT only. This positive therapeutic result lasted for at least 6 months after the end of the treatment. These findings support the combined use of EPI® and APT to treat ALErGP.

Therapeutic results after ultrasound-guided intratissue percutaneous electrolysis (EPI®) in the treatment of rectus abdominis-related groin pain in professional footballers: a pilot study.

BACKGROUND: Rectus abdominis-related groin pain (RAGP) is one of the possible clinical patterns that determine pubalgia. RAGP is one of the typical patterns in footballers and is due to the degeneration and tendinopathy of the distal tendon, at the level of the two pubic tubercles. Intratissue percutaneous electrolysis (EPI) is a novel technique used in the treatment of tendinopathies. The aim of this study was to examine the therapeutic benefits of EPI by contrasting the two basic components that characterize RAGP: painful symptoms and resultant functional deficits. METHODS: Eight professional footballers at Udinese Calcio Spa Football Club underwent ultrasound-guided EPI intervention. No other type of treatment was combined with EPI. Pain was monitored with the Verbal Rating Scale, while functional deficit was monitored using the Patient Specific Functional Scale. The scales implementation took place before treatment, then 24 hours, 1 week, 1 month and 6 months after the end of treatment. RESULTS: Treatment with EPI produced a complete reduction of pain symptoms in one month and enabled excellent functional recovery for walking and jogging in one week; for getting out of bed, running, jumping and kicking within one month from the end of the treatment. CONCLUSIONS: Treatment with ultrasound-guided EPI has shown encouraging clinical results for RAGP. Data are preliminary: considering the limitations of this study more complex design studies are necessary to test the efficacy of the technique. This study introduces the EPI technique for the first time in the treatment of professional footballers suffering from RAGP. Its future use is proposed as a treatment solution, including complementary to conservative treatment.

Running demands and heart rate responses in men Rugby Sevens.

The purpose of this study was to examine match running performance and exercise intensity in a Rugby Sevens (7s) team during competitive club-level matches. Time-motion analyses (global position system) were performed on 7 male rugby players during 5 competitive matches in a 2-day tournament. The players covered an average distance of 1,580.8 ± 146.3 m per game (14 minutes). Over this distance, 34.8% (549.7 ± 79.1 m) was spent standing and walking, 26.2% (414.8 ± 105.1 m) jogging, 9.8% (154.6 ± 53.5 m) cruising, 15.5% (244.5 ± 80.1 m) striding, 5% (79.5 ± 37.2 m) high-intensity running, and 8.7% (137.7 ± 84.9 m) sprinting. The average maximal distance of sprints, the number of sprints, the minimum distance of sprint, and the mean sprint distance over the game were 29.5 ± 11.7 m, 7.4 ± 3.9 sprints, 9.1 ± 5.7 m, and 18 ± 7.6 m, respectively. The player's work-to-rest ratio was 1:0.5. For over 75% of the game, the players were exposed to heart rates (HRs) >80% of their maximal HR. There were no statistical differences between the first and second halves in any of the variables analyzed. This study indicates that the physical demands of Rugby 7s are quite different from those encountered in other rugby codes and that the training regimes need to meet the increased overall running demands, the augmented high-intensity running actions, and the reduced work-to-rest ratios.

Match running performance and exercise intensity in elite female Rugby Sevens.

The purpose of this study was to describe the match-play demands of professional female rugby players competing in Rugby Sevens (Rugby 7's) matches. Time-motion analyses (global position system) were performed on 12 elite female rugby players during 5 competitive matches in a 2-day international tournament. Data revealed that players covered an average distance of 1,556.2 ± 189.3 m per game (14 minutes). Over this distance, 29.7% (462.6 ± 94.6 m) was spent standing and walking, 33.2% (515.9 ± 88.6 m) jogging, 11.6% (181.0 ± 61.4 m) cruising, 16.4% (255.7 ± 88.3 m) striding, 3.7% (57.1 ± 40.8 m) high-intensity running, and 5.4% (84.0 ± 64.8 m) sprinting. The average maximal distance of sprints, number of sprints, minimum distance of sprint, and mean sprint distance over the game were as follows: 25.8 ± 16.1 m, 5.3 ± 3.2 sprints, 6.5 ± 2.0 m, and 17.2 ± 8.8 m, respectively. The players' work-to-rest ratio was 1:0.4. For over 75% of the game, the players were exposed to heart rates (HRs) >80% of their maximal HR. There were no statistical differences between the first and second halves in any of the variables analyzed. This study suggests that the physical demands of Rugby 7's are quite different from those reported in other rugby codes. For players and teams to remain competitive in female Rugby Sevens, coaching, conditioning, and physical fitness testing should reflect these current demands.

Emergency visits for childhood poisoning: a 2-year prospective multicenter survey in Spain.

OBJECTIVES: To describe the characteristics of childhood poisoning leading to consultation to 17 pediatric emergency departments in Spain. METHODS: During a 2-year period (January 2001 to December 2002), accompanying people of 2157 children with acute intoxication who visited consecutively at the emergency room were prospectively surveyed. RESULTS: Childhood poisoning accounted for 0.28% of all emergency visits during the study period. The median (interquartile range, 25th-75th percentile) age was 24 months (22-60 months); 67% of children were younger than 4 years. Drug ingestion was involved in 54.7% of cases (paracetamol was the most frequent drug), domestic products in 28.9%, alcohol in 5.9%, carbon monoxide in 4.5%, and illicit drugs in 1.5%. A total of 61.3% of patients were admitted within 1 hour after exposure to the toxic substance, and 10.3% had been already treated before arrival; 29.1% of patients were referred for clinical manifestations which were mostly neurological symptoms. Laboratory tests and other investigations were performed in 40.7% of cases. Gastrointestinal decontamination was used in 51.7% of patients, with activated charcoal in 32.3%. Treatment varied significantly according to the individual hospitals. A total of 83.3% of patients were treated as outpatients, 15.2% were hospitalized, and 1.5% were admitted to the intensive care unit. One 11-month-old boy with carbon monoxide intoxication died. Six patients had permanent sequelae (esophageal stenosis in 5 and partial blindness in 1). CONCLUSIONS: Young children who accidentally ingested drugs and, less frequently, domestic products accounted for most cases of intoxication who presented at the pediatric emergency department.