Mechanical stimulation-induced purinome priming fosters osteogenic differentiation and osteointegration of mesenchymal stem cells from the bone marrow of post-menopausal women.

BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model. METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation. RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model. CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.

2,4,5-Triaminopyrimidines as blue fluorescent probes for cell viability monitoring: synthesis, photophysical properties, and microscopy applications.

Monitoring cell viability is critical in cell biology, pathology, and drug discovery. Most cell viability assays are cell-destructive, time-consuming, expensive, and/or hazardous. Herein, we present a series of newly synthesized 2,4,5-triaminopyrimidine derivatives able to discriminate between live and dead cells. To our knowledge, these compounds are the first fluorescent nucleobase analogues (FNAs) with cell viability monitoring potential. These new fluorescent molecules are synthesized using highly efficient and cost-effective methods and feature unprecedented photophysical properties (longer absorption and emission wavelengths, environment-sensitive emission, and unprecedented brightness within FNAs). Using a live-dead Saccharomyces cerevisiae cell and theoretical assays, the fluorescent 2,4,5-triaminopyrimidine derivatives were found to specifically accumulate inside dead cells by interacting with dsDNA grooves, thus paving the way for the emergence of novel and safe fluorescent cell viability markers emitting in the blue region. As the majority of commercially available viability dyes emit in the green to red region of the visible spectrum, these novel markers might be useful to meet the needs of blue markers for co-staining combinations.

Blockage of the adenosine A2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension.

Sustained pressure overload and fibrosis of the right ventricle (RV) are the leading causes of mortality in pulmonary arterial hypertension (PAH). Although the role of adenosine in PAH has been attributed to the control of pulmonary vascular tone, cardiac reserve, and inflammatory processes, the involvement of the nucleoside in RV remodelling remains poorly understood. Conflicting results exist on targeting the low-affinity adenosine A2B receptor (A2BAR) for the treatment of PAH mostly because it displays dual roles in acute vs. chronic lung diseases. Herein, we investigated the role of the A2BAR in the viability/proliferation and collagen production by cardiac fibroblasts (CFs) isolated from RVs of rats with monocrotaline (MCT)-induced PAH. CFs from MCT-treated rats display higher cell viability/proliferation capacity and overexpress A2BAR compared to the cells from healthy littermates. The enzymatically stable adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA, 1-30 µM), concentration-dependently increased growth, and type I collagen production by CFs originated from control and PAH rats, but its effects were more prominent in cells from rats with PAH. Blockage of the A2BAR with PSB603 (100 nM), but not of the A2AAR with SCH442416 (100 nM), attenuated the proliferative effect of NECA in CFs from PAH rats. The A2AAR agonist, CGS21680 (3 and 10 nM), was virtually devoid of effect. Overall, data suggest that adenosine signalling via A2BAR may contribute to RV overgrowth secondary to PAH. Therefore, blockage of the A2AAR may be a valuable therapeutic alternative to mitigate cardiac remodelling and prevent right heart failure in PAH patients.

A comprehensive transcriptomic analysis of the bisphenol A affected kidney in mice.

Introduction: Bisphenol A (BPA) is a substance belonging to the endocrine-disrupting chemicals, globally used in the production of polycarbonate plastics. It has been found that BPA enhances carcinogenesis, triggers obesity and exerts a pathogenic effect in several disorders, such as type 2 diabetes, asthma, or increased blood pressure. Recent studies have revealed, that BPA has a harmful impact on the kidneys function, therefore, the current research aimed to explore the specific molecular changes triggered in these organs after oral BPA exposure in mice. Materials and Methods: The experiment was carried out on 12 (3-month-old) female mice. Six mice served as controls. The other 6 mice were treated with BPA in the drinking water at a dose of 50 mg/kg b. w. for 3 months. Then animals were euthanized, the kidneys were collected, and extracted RNA was used to perform RNA-seq. Results: Applied multistep bioinformatics revealed 433 differentially expressed genes (DEGs) in the BPA-treated kidneys (232 upregulated and 201 downregulated). Additionally, 95 differentially expressed long-noncoding RNAs (DELs) were revealed in BPA samples. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations indicated that BPA exposure resulted in profound changes in several essential processes, such as oxidative phosphorylation, mitochondrial and ribosome function, or chemical carcinogenesis. Conclusion: The obtained novel results suggest that BPA has a harmful impact on the fundamental processes of the kidney and significantly impairs its function by inducing mitochondrial dysfunction leading to oxidative stress and reactive oxygen species production.

Adenosinergic System and BDNF Signaling Changes as a Cross-Sectional Feature of RTT: Characterization of Mecp2 Heterozygous Mouse Females.

Rett Syndrome is an X-linked neurodevelopmental disorder (RTT; OMIM#312750) associated to MECP2 mutations. MeCP2 dysfunction is seen as one cause for the deficiencies found in brain-derived neurotrophic factor (BDNF) signaling, since BDNF is one of the genes under MeCP2 jurisdiction. BDNF signaling is also dependent on the proper function of the adenosinergic system. Indeed, both BDNF signaling and the adenosinergic system are altered in Mecp2-null mice (Mecp2-/y), a representative model of severe manifestation of RTT. Considering that symptoms severity largely differs among RTT patients, we set out to investigate the BDNF and ADO signaling modifications in Mecp2 heterozygous female mice (Mecp2+/-) presenting a less severe phenotype. Symptomatic Mecp2+/- mice have lower BDNF levels in the cortex and hippocampus. This is accompanied by a loss of BDNF-induced facilitation of hippocampal long-term potentiation (LTP), which could be restored upon selective activation of adenosine A2A receptors (A2AR). While no differences were observed in the amount of adenosine in the cortex and hippocampus of Mecp2+/- mice compared with healthy littermates, the density of the A1R and A2AR subtype receptors was, respectively, upregulated and downregulated in the hippocampus. Data suggest that significant changes in BDNF and adenosine signaling pathways are present in an RTT model with a milder disease phenotype: Mecp2+/- female animals. These features strengthen the theory that boosting adenosinergic activity may be a valid therapeutic strategy for RTT patients, regardless of their genetic penetrance.

Putative Identification of New Phragmaline-Type Limonoids from the Leaves of Swietenia macrophylla King: A Case Study Using Mass Spectrometry-Based Molecular Networking.

Swietenia macrophylla King is a plant commonly known as Brazilian mahogany. The wood from its stem is highly prized for its exceptional quality, while its leaves are valued for their high content of phragmalin-type limonoids, a subclass of compounds known for their significant biological activities, including antimalarial, antitumor, antiviral, and anti-inflammatory properties. In this context, twelve isolated limonoids from S. macrophylla leaves were employed as standards in mass spectrometry-based molecular networking to unveil new potential mass spectrometry signatures for phragmalin-type limonoids. Consequently, ultra-performance liquid chromatography coupled with high-resolution mass spectrometry was utilized for data acquisition. Subsequently, the obtained data were analyzed using the Global Natural Products Social Molecular Networking platform based on spectral similarity. In summary, this study identified 24 new putative phragmalin-type limonoids for the first time in S. macrophylla. These compounds may prove valuable in guiding future drug development efforts, leveraging the already established biological activities associated with limonoids.

Delphi consensus on add-ons and social midia in Assisted Reproductive Technology.

This article reports the annals of a national consensus meeting on add-ons and social networks in Assisted Reproduction Techniques (ART). The panel of experts has developed a set of consensus points and this document is intended to be referenced as a national consensus to allow social networks and add-ons to be used in ART, following the standards of the Code of Medical Ethics and the Federal Council of Medicine, in a safe ethical and responsible way.

The Carcinogenic Potential of Bisphenol A in the Liver Based on Transcriptomic Studies.

Bisphenol A (BPA) is an environmental toxin widely used in the production of polycarbonate plastics. A correlation exists between BPA tissue contamination and the occurrence of pathological conditions, including cancer. First-passage detoxification of high BPA amounts in the liver promotes hepatotoxicity and morphological alterations of this organ, but there is a lack of knowledge about the molecular mechanisms underlying these phenomena. This prompted us to investigate changes in the liver transcriptomics of 3-month-old female mice exposed to BPA (50 mg/kg) in drinking water for 3 months. Five female mice served as controls. The animals were euthanized, the livers were collected, and RNA was extracted to perform RNA-seq analysis. The multistep transcriptomic bioinformatics revealed 120 differentially expressed genes (DEGs) in the BPA-exposed samples. Gene Ontology (GO) annotations indicated that DEGs have been assigned to many biological processes, including "macromolecule modification" and "protein metabolic process". Several of the revealed DEGs have been linked to the pathogenesis of severe metabolic liver disorders and malignant tumors, in particular hepatocellular carcinoma. Data from this study suggest that BPA has a significant impact on gene expression in the liver, which is predictive of the carcinogenic potential of this compound in this organ.

Could hypoxia rehabilitate the osteochondral diseased interface? Lessons from the interplay of hypoxia and purinergic signals elsewhere.

The osteochondral unit comprises the articular cartilage (90%), subchondral bone (5%) and calcified cartilage (5%). All cells present at the osteochondral unit that is ultimately responsible for matrix production and osteochondral homeostasis, such as chondrocytes, osteoblasts, osteoclasts and osteocytes, can release adenine and/or uracil nucleotides to the local microenvironment. Nucleotides are released by these cells either constitutively or upon plasma membrane damage, mechanical stress or hypoxia conditions. Once in the extracellular space, endogenously released nucleotides can activate membrane-bound purinoceptors. Activation of these receptors is fine-tuning regulated by nucleotides' breakdown by enzymes of the ecto-nucleotidase cascade. Depending on the pathophysiological conditions, both the avascular cartilage and the subchondral bone subsist to significant changes in oxygen tension, which has a tremendous impact on tissue homeostasis. Cell stress due to hypoxic conditions directly influences the expression and activity of several purinergic signalling players, namely nucleotide release channels (e.g. Cx43), NTPDase enzymes and purinoceptors. This review gathers experimental evidence concerning the interplay between hypoxia and the purinergic signalling cascade contributing to osteochondral unit homeostasis. Reporting deviations to this relationship resulting from pathological alterations of articular joints may ultimately unravel novel therapeutic targets for osteochondral rehabilitation. At this point, one can only hypothesize how hypoxia mimetic conditions can be beneficial to the ex vivo expansion and differentiation of osteo- and chondro-progenitors for auto-transplantation and tissue regenerative purposes.

Silencing NTPDase3 activity rehabilitates the osteogenic commitment of post-menopausal stem cell bone progenitors.

BACKGROUND: Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs. METHODS: MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC RESULTS: The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y6 purinoceptors prevented this effect. CONCLUSIONS: Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y6 receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.

Purinergic Tuning of the Tripartite Neuromuscular Synapse.

The vertebrate neuromuscular junction (NMJ) is a specialised chemical synapse involved in the transmission of bioelectric signals between a motor neuron and a skeletal muscle fiber, leading to muscle contraction. Typically, the NMJ is a tripartite synapse comprising (a) a presynaptic region represented by the motor nerve ending, (b) a postsynaptic skeletal motor endplate area, and (c) perisynaptic Schwann cells (PSCs) that shield the motor nerve terminal. Increasing evidence points towards the role of PSCs in the maintenance and control of neuromuscular integrity, transmission, and plasticity. Acetylcholine (ACh) is the main neurotransmitter at the vertebrate skeletal NMJ, and its role is fine-tuned by co-released purinergic neuromodulators, like adenosine 5'-triphosphate (ATP) and its metabolite adenosine (ADO). Adenine nucleotides modulate transmitter release and expression of postsynaptic ACh receptors at motor synapses via the activation of P2Y and P2X receptors. Endogenously generated ADO modulates ACh release by acting via co-localised inhibitory A1 and facilitatory A2A receptors on motor nerve terminals, whose tonic activation depends on the neuronal firing pattern and their interplay with cholinergic receptors and neuropeptides. Thus, the concerted action of adenine nucleotides, ADO, and ACh/neuropeptide co-transmitters is paramount to adapting the neuromuscular transmission to the working load under pathological conditions, like Myasthenia gravis. Unravelling these functional complexities prompted us to review our knowledge about the way purines orchestrate neuromuscular transmission and plasticity in light of the tripartite synapse concept, emphasising the often-forgotten role of PSCs in this context.

Counteracting Colon Cancer by Inhibiting Mitochondrial Respiration and Glycolysis with a Selective PKCδ Activator.

Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.

RNA-seq reveals that anti-obesity irisin and triiodothyronine (T3) hormones differentially affect the purinergic signaling transcriptomics in differentiated human adipocytes.

The anti-obesity thyroid hormone, triiodothyronine (T3), and irisin, an exercise- and/or cold-induced myokine, stimulate thermogenesis and energy consumption while decreasing lipid accumulation. The involvement of ATP signaling in adipocyte cell function and obesity has attracted increasing attention, but the crosstalk between the purinergic signaling cascade and anti-obesity hormones lacks experimental evidence. In this study, we investigated the effects of T3 and irisin in the transcriptomics of membrane-bound purinoceptors, ectonucleotidase enzymes and nucleoside transporters participating in the purinergic signaling in cultured human adipocytes. The RNA-seq analysis revealed that differentiated adipocytes express high amounts of ADORA1, P2RY11, P2RY12, and P2RX6 gene transcripts, along with abundant levels of transcriptional products encoding to purine metabolizing enzymes (ENPP2, ENPP1, NT5E, ADA and ADK) and transporters (SLC29A1, SCL29A2). The transcriptomics of purinergic signaling markers changed in parallel to the upsurge of "browning" adipocyte markers, like UCP1 and P2RX5, after treatment with T3 and irisin. Upregulation of ADORA1, ADORA2A and P2RX4 gene transcription was obtained with irisin, whereas T3 preferentially upregulated NT5E, SLC29A2 and P2RY11 genes. Irisin was more powerful than T3 towards inhibition of the leptin gene transcription, the SCL29A1 gene encoding for the ENT1 transporter, the E-NPP2 (autotaxin) gene, and genes that encode for two ADP-sensitive P2Y receptors, P2RY1 and P2RY12. These findings indicate that anti-obesity irisin and T3 hormones differentially affect the purinergic signaling transcriptomics, which might point towards new directions for the treatment of obesity and related metabolic disorders that are worth to be pursued in future functional studies.

Molecular pathways underlying sympathetic autonomic overshooting leading to fear and traumatic memories: looking for alternative therapeutic options for post-traumatic stress disorder.

The sympathoadrenal medullary system and the hypothalamic-pituitary-adrenal axis are both activated upon stressful events. The release of catecholamines, such as dopamine, norepinephrine (NE), and epinephrine (EPI), from sympathetic autonomic nerves participate in the adaptive responses to acute stress. Most theories suggest that activation of peripheral ß-adrenoceptors (ß-ARs) mediates catecholamines-induced memory enhancement. These include direct activation of ß-ARs in the vagus nerve, as well as indirect responses to catecholamine-induced glucose changes in the brain. Excessive sympathetic activity is deeply associated with memories experienced during strong emotional stressful conditions, with catecholamines playing relevant roles in fear and traumatic memories consolidation. Recent findings suggest that EPI is implicated in fear and traumatic contextual memories associated with post-traumatic stress disorder (PTSD) by increasing hippocampal gene transcription (e.g., Nr4a) downstream to cAMP response-element protein activation (CREB). Herein, we reviewed the literature focusing on the molecular mechanisms underlying the pathophysiology of memories associated with fear and traumatic experiences to pave new avenues for the treatment of stress and anxiety conditions, such as PTSD.

Remote Heart Rate Prediction in Virtual Reality Head-Mounted Displays Using Machine Learning Techniques.

Head-mounted displays are virtual reality devices that may be equipped with sensors and cameras to measure a patient's heart rate through facial regions. Heart rate is an essential body signal that can be used to remotely monitor users in a variety of situations. There is currently no study that predicts heart rate using only highlighted facial regions; thus, an adaptation is required for beats per minute predictions. Likewise, there are no datasets containing only the eye and lower face regions, necessitating the development of a simulation mechanism. This work aims to remotely estimate heart rate from facial regions that can be captured by the cameras of a head-mounted display using state-of-the-art EVM-CNN and Meta-rPPG techniques. We developed a region of interest extractor to simulate a dataset from a head-mounted display device using stabilizer and video magnification techniques. Then, we combined support vector machine and FaceMash to determine the regions of interest and adapted photoplethysmography and beats per minute signal predictions to work with the other techniques. We observed an improvement of 188.88% for the EVM and 55.93% for the Meta-rPPG. In addition, both models were able to predict heart rate using only facial regions as input. Moreover, the adapted technique Meta-rPPG outperformed the original work, whereas the EVM adaptation produced comparable results for the photoplethysmography signal.

A2A receptor-induced overexpression of pannexin-1 channels indirectly mediates adenosine fibrogenic actions by favouring ATP release from human subcutaneous fibroblasts.

AIMS: Disorganization of the subcutaneous tissue due to inflammation and fibrosis is a common feature in patients with myofascial pain. Dermal accumulation of adenosine favours collagen production by human subcutaneous fibroblasts (HSCF) via A2A receptors (A2AR) activation. Adenosine mimics the fibrogenic effect of inflammatory mediators (e.g. histamine, bradykinin), which promote ATP release from HSCF via plasma-membrane-bound pannexin-1 (Panx1) and/or connexin-43 (Cx43) channels, but this mechanism has never been implicated in A2AR actions. MATERIALS AND METHODS: A2AR-mediated effects on Panx1 and Cx43 protein amounts were evaluated in primary cultures of HSCF by confocal microscopy and Western blot analysis. Functional repercussions in collagen production, intracellular [Ca2+]i oscillations and ATP release were also evaluated. KEY FINDINGS: NECA and CGS21680, two enzymatically-stable A2AR agonists, increased Panx1, but reduced Cx43, protein density in HSCF. This effect was accompanied by increases in ATP release and collagen III production by HSCF. The involvement of the A2AR was confirmed by blockage with the selective A2AR antagonist, SCH442416. Inhibition of Panx1 channels by probenecid and the Panx1 mimetic inhibitory peptide, 10Panx, also decreased ATP release and collagen production by HSCF under similar conditions. Superfluous ATP release by HSCF exposed to A2AR agonists overexpressing Panx1 channels contributes to keeping high [Ca2+]i levels when the cells were exposed to histamine. SIGNIFICANCE: Adenosine A2AR-induced Panx1 overexpression was shown here for the first time in HSCF; this feature indirectly implicates ATP release in the fibrogenic vicious cycle operated by adenosine accumulating in subcutaneous tissue fibrosis and myofascial pain associated to dermal inflammation.

Mesial Temporal Lobe Epilepsy (MTLE) Drug-Refractoriness Is Associated With P2X7 Receptors Overexpression in the Human Hippocampus and Temporal Neocortex and May Be Predicted by Low Circulating Levels of miR-22.

Objective: ATP-gated ionotropic P2X7 receptors (P2X7R) actively participate in epilepsy and other neurological disorders. Neocortical nerve terminals of patients with Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS) express higher P2X7R amounts. Overexpression of P2X7R bolsters ATP signals during seizures resulting in glial cell activation, cytokines production, and GABAergic rundown with unrestrained glutamatergic excitation. In a mouse model of status epilepticus, increased expression of P2X7R has been associated with the down-modulation of the non-coding micro RNA, miR-22. MiR levels are stable in biological fluids and normally reflect remote tissue production making them ideal disease biomarkers. Here, we compared P2X7R and miR-22 expression in epileptic brains and in the serum of patients with MTLE-HS, respectively. Methods: Quantitative RT-PCR was used to evaluate the expression of P2X7R in the hippocampus and anterior temporal lobe of 23 patients with MTLE-HS and 10 cadaveric controls. Confocal microscopy and Western blot analysis were performed to assess P2X7R protein amounts. MiR-22 expression was evaluated in cell-free sera of 40 MTLE-HS patients and 48 healthy controls. Results: Nerve terminals of the hippocampus and neocortical temporal lobe of MTLE-HS patients overexpress (p < 0.05) an 85 kDa P2X7R protein whereas the normally occurring 67 kDa receptor protein dominates in the brain of the cadaveric controls. Contrariwise, miR-22 serum levels are diminished (p < 0.001) in MTLE-HS patients compared to age-matched control blood donors, a situation that is more evident in patients requiring multiple (>3) anti-epileptic drug (AED) regimens. Conclusion: Data show that there is an inverse relationship between miR-22 serum levels and P2X7R expression in the hippocampus and neocortex of MTLE-HS patients, which implies that measuring serum miR-22 may be a clinical surrogate of P2X7R brain expression in the MTLE-HS. Moreover, the high area under the ROC curve (0.777; 95% CI 0.629-0.925; p = 0.001) suggests that low miR-22 serum levels may be a sensitive predictor of poor response to AEDs among MTLE-HS patients. Results also anticipate that targeting the miR-22/P2X7R axis may be a good strategy to develop newer AEDs.

Margaritaria nobilis L.F. (Phyllanthaceae): Ethnopharmacology and Application of Computational Tools in the Annotation of Bioactive Molecules.

Margaritaria nobilis is a shrubby species widely distributed in Brazil from the Amazon to the Atlantic Rainforest. Its bark and fruit are used in the Peruvian Amazon for disinfecting abscesses and as a tonic in pregnancy, respectively, and its leaves are used to treat cancer symptoms. From analyses via UHPLC-MS/MS, we sought to determine the chemical profile of the ethanolic extract of M. nobilis leaves by means of putative analyses supported by computational tools and spectral libraries. Thus, it was possible to annotate 44 compounds, of which 12 are phenolic acid derivatives, 16 are O-glycosylated flavonoids and 16 hydrolysable tannins. Among the flavonoids, although they are known, except for kaempferol, which has already been isolated from this species, the other flavonoids (10, 14, 15, 21, 24-26, 28-30, 33-35, 40 and 41) are being reported for the first time in the genus. Among the hydrolysable tannins, six ellagitannins present the HHDP group (6, 19, 22, 31, 38 and 43), one presents the DHHDP group (5), and four contain oxidatively modified congeners (12, 20, 37 and 39). Through the annotation of these compounds, we hope to contribute to the improved chemosystematics knowledge of the genus. Furthermore, supported by a metric review of the literature, we observed that many of the compounds reported here are congeners of authentically bioactive compounds. Thus, we believe that this work may help in understanding future pharmacological activities.

Pharmacological Tuning of Adenosine Signal Nuances Underlying Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) roughly represents half of the cardiac failure events in developed countries. The proposed 'systemic microvascular paradigm' has been used to explain HFpHF presentation heterogeneity. The lack of effective treatments with few evidence-based therapeutic recommendations makes HFpEF one of the greatest unmet clinical necessities worldwide. The endogenous levels of the purine nucleoside, adenosine, increase significantly following cardiovascular events. Adenosine exerts cardioprotective, neuromodulatory, and immunosuppressive effects by activating plasma membrane-bound P1 receptors that are widely expressed in the cardiovascular system. Its proven benefits have been demonstrated in preclinical animal tests. Here, we provide a comprehensive and up-to-date critical review about the main therapeutic advantages of tuning adenosine signalling pathways in HFpEF, without discounting their side effects and how these can be seized.