Uracil Research Articles

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

BackgroundMechanical 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.MethodsBM-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.ResultsMS-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.ConclusionsData 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.

Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.

Characterization of a FourU RNA Thermometer in the 5' Untranslated Region of Autolysin Gene blyA in the Bacillus subtilis 168 Prophage SPβ.

RNA thermometers are noncoding RNA structures located in the 5' untranslated regions (UTRs) of genes that regulate gene expression through temperature-dependent conformational changes. The fourU class of RNA thermometers contains a specific motif in which four consecutive uracil nucleotides are predicted to base pair with the Shine-Dalgarno (SD) sequence in a stem. We employed a bioinformatic search to discover a fourU RNA thermometer in the 5'-UTR of the blyA gene of the Bacillus subtilis phage SPβc2, a bacteriophage that infects B. subtilis 168. blyA encodes an autolysin enzyme, N-acetylmuramoyl-l-alanine amidase, which is involved in the lytic life cycle of the SPβ prophage. We have biochemically validated the predicted RNA thermometer in the 5'-UTR of the blyA gene. Our study suggests that RNA thermometers may play an underappreciated yet critical role in the lytic life cycle of bacteriophages.

The first nano-cocrystal formulation of marine drug cytarabine with uracil based on cocrystal nanonization strategy for long-acting injection exhibiting enhanced antitumor activity

To emphasize the superiority of uracil (UR) in ameliorating biopharmaceutical characteristics of marine antitumor medicine cytarabine (ARA), thus gaining some innovative opinions for the exploitation of nanococrystal formulation, a cocrystal nanonization strategy is proposed by integrating cocrystallization and nanosize preparation techniques. For one thing, based on UR's unique structural features and natures together with advantages of preferential uptake by tumor cells, cocrystallizing ARA with UR is expected to improve the in vitro/vivo performances. For another, the nanonization procedure is oriented towards maintaining the long-term effective drug level. Along this route, a cocrystal of ARA with UR, viz., ARA-UR, is successfully synthesized and then transformed into nano-cocrystal. The cocrystal structure is precisely confirmed by various methods, demonstrating that a 1:1 ARA and UR in the crystal forms cytosine-UR hydrogen-bonding interactions, thus constructing supramolecular frameworks by strong π-π stacking interplays; while the nano-cocrystal is block-shaped particles of 562.70 nm with zeta potential −33.40 mV. The properties of cocrystal ARA-UR and its nano-cocrystal in vitro/vivo are comparatively explored by theoretical calculations and experimental analyses, revealing that permeability of both is significantly increased than ARA per se. Notably, the meliorative natures of both the cocrystal and nano-cocrystal in vitro bring excellent antitumor activity, but the latter has greater strengths over the former. More notably, the nano-cocrystal can sustain effective concentration for a relatively longer time, causing lengthened retention time and better absorption in vivo. The contribution offers a fire-new dosage form of ARA for long-lasting delivery, thus filling the vacancy in nanococrystal studies about marine drugs.

Quenching mechanism of PEG emission by pyrimidines and their detections using PEG as fluorescent sensor

Pyrimidines, in particular uracil (UL) and 5-fluorouracil (5FU), are well known anticancerogens and widely used against a variety of cancers. An important concerning issue has been their monitoring and detection in order to adequately control their doses and to minimize their health hazards. Among a variety of methods developed, fluorescence sensing is gaining more attention because of its accuracy, easy operation, and straight visuability. However, in most of the reported studies, the sensors are made from transition metals with sophisticated multi-processes; polymers, particularly biopolymers, are hardly available. We demonstrate herein that aqueous solution of poly(ethylene glycol) (PEG), a commonly known biopolymer, is highly emissive under excitation of 280 nm. While the solutions of selected pyrimidines, i.e. UL and 5FU, are non-emissive under the same excitation, their addition to PEG solution, however, brings about a distinct and concentration dependent quenching for PEG emission. PEG was then used as sensor with good stability for UL and 5FU detections in water. The tests were also done by spiking 5FU in tap water, milk and synthetic urine, confirming that 5FU was practically fully recovered in all the cases. The quenching was ascribed to the interaction between PEG clusters and the heteroatoms on pyrimidine molecules through hydrogen bonding, which is supported by multi-technical analyses, including NMR, FTIR and cluster size tests by DLS. This work provides therefore a full green, easy to operate, low-costing, novel and reliable protocol for pyrimidine monitoring and detection, with the quenching mechanism interpreted based on multi-technical characterizations.

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

BackgroundEndogenously 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.MethodsMSCs 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 HPLCResultsThe 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.ConclusionsData 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.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Extracellular adenosine 5ʹ-diphosphate promotes MCP-1/CCL2 expression via the P2Y13 purinergic receptor/ERK signaling axis in temporomandibular joint-derived mouse fibroblast-like synoviocytes

BackgroundTemporomandibular joint osteoarthritis (TMJ-OA) causes cartilage degeneration, bone cavitation, and fibrosis of the TMJ. However, the mechanisms underlying the fibroblast-like synoviocyte (FLS)-mediated inflammatory activity in TMJ-OA remain unclear.Methods and resultsReverse transcription-quantitative polymerase chain reaction analysis revealed that the P2Y1, P2Y12, and P2Y13 purinergic receptor agonist adenosine 5ʹ-diphosphate (ADP) significantly induces monocyte chemotactic protein 1 (MCP-1)/ C–C motif chemokine ligand 2 (CCL2) expression in the FLS1 synovial cell line. In contrast, the uracil nucleotide UTP, which is a P2Y2 and P2Y4 agonist, has no significant effect on MCP-1/CCL2 production in FLS1 cells. In addition, the P2Y13 antagonist MRS 2211 considerably decreases the expression of ADP-induced MCP-1/CCL2, whereas ADP stimulation enhances extracellular signal-regulated kinase (ERK) phosphorylation. Moreover, it was found that the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor U0126 reduces ADP-induced MCP-1/CCL2 expression.ConclusionADP enhances MCP-1/CCL2 expression in TMJ FLSs via P2Y13 receptors in an MEK/ERK-dependent manner, thus resulting in inflammatory cell infiltration in the TMJ. Collectively, the findings of this study contribute to a partial clarification of the signaling pathway underlying the development of inflammation in TMJ-OA and can help identify potential therapeutic targets for suppressing ADP-mediated purinergic signaling in this disease.

Chemistry of glycol nucleic acid (GNA): Synthesis, photophysical characterization and insight into the biological activity of phenanthrenyl GNA constituents

The knowledge pertaining to the chemistry and biological activity of glycol nucleic acid (GNA) components, like nucleosides and nucleotides, is still very limited. Herein we report on the preparation of the uracil nucleoside (1) and nucleotide ester GNA (2). The compounds are functionalised with a luminescent phenanthrenyl group. In DMSO, 1 and 2 are brightly fluorescent, with emission maxima at 390 nm, nanosecond decay times (0.6 and 0.75 ns, respectively), and quantum yields of ca. 0.2. In the solid phase, they show excimeric emission, with maxima at 495 nm (1) and 432 nm (2), and decay times of 3.7 ns (1) and 2.9 ns (2). The anticancer activity of the GNA components, as well as gemcitabine hydrochloride, used as a reference drug, were examined in vitro against human cancer HeLa and Ishikawa cells, as well as against normal L929 cells, using a battery of biochemical assays. Furthermore, biodistribution imaging studies were carried out in HeLa cells, with luminescence confocal microscopy, which showed that the compounds localized mainly in the lipophilic cellular compartments. Nucleoside (1) and nucleotide ester (2) features two different anticancer activity profiles. At 24 h of treatment, the nucleoside acts mainly as a toxin and induces necrosis in HeLa cells, whereas the nucleotide ester exhibits pro-apoptotic activity. At longer treatment times (72 h), the nucleoside and the reference, gemcitabine hydrochloride, featured almost identical signs of anticancer activity, such as S-phase cell cycle arrest, proliferation inhibition, and apoptosis induction. In view of this data, one can hypothesize that despite the structural differences, the newly obtained phenanthrenyl GNA nucleoside (1) and gemcitabine may share a common mechanism of anticancer activity in HeLa cancer cells. The GNA components were also examined as antiplasmodial agents against Plasmodium falciparum, in vitro. Nucleoside (1) was found to be more potent than nucleotide (2), displaying activity in the low micromolar range. Furthermore, both phenanthrene derivatives were found to display resistance indices at least 9-fold lower than chloroquine diphosphate (CQDP).

Predicting Drosha and Dicer Cleavage Sites with DeepMirCut.

MicroRNAs are a class of small RNAs involved in post-transcriptional gene silencing with roles in disease and development. Many computational tools have been developed to identify novel microRNAs. However, there have been no attempts to predict cleavage sites for Drosha from primary sequence, or to identify cleavage sites using deep neural networks. Here, we present DeepMirCut, a recurrent neural network-based software that predicts both Dicer and Drosha cleavage sites. We built a microRNA primary sequence database including flanking genomic sequences for 34,713 microRNA annotations. We compare models trained on sequence data, sequence and secondary structure data, as well as input data with annotated structures. Our best model is able to predict cuts within closer average proximity than results reported for other methods. We show that a guanine nucleotide before and a uracil nucleotide after Dicer cleavage sites on the 3′ arm of the microRNA precursor had a positive effect on predictions while the opposite order (U before, G after) had a negative effect. Our analysis was also able to predict several positions where bulges had either positive or negative effects on the score. We expect that our approach and the data we have curated will enable several future studies.

Evolution of Functionally Enhanced α-l-Threofuranosyl Nucleic Acid Aptamers.

Synthetic genetic polymers (xeno-nucleic acids, XNAs) have the potential to transition aptamers from laboratory tools to therapeutic agents, but additional functionality is needed to compete with antibodies. Here, we describe the evolution of a biologically stable artificial genetic system composed of α-l-threofuranosyl nucleic acid (TNA) that facilitates the production of backbone- and base-modified aptamers termed "threomers" that function as high quality protein capture reagents. Threomers were discovered against two prototypical protein targets implicated in human diseases through a combination of in vitro selection and next-generation sequencing using uracil nucleotides that are uniformly equipped with aromatic side chains commonly found in the paratope of antibody-antigen crystal structures. Kinetic measurements reveal that the side chain modifications are critical for generating threomers with slow off-rate binding kinetics. These findings expand the chemical space of evolvable non-natural genetic systems to include functional groups that enhance protein target binding by mimicking the structural properties of traditional antibodies.

Molecular modeling and dynamics studies of the synthetic small molecule agonists with GPR17 and P2Y1 receptor

The human Guanine Protein coupled membrane Receptor 17 (hGPR17), an orphan receptor that activates uracil nucleotides and cysteinyl leukotrienes is considered as a crucial target for the neurodegenerative diseases. Yet, the detailed molecular interaction of potential synthetic ligands of GPR17 needs to be characterized. Here, we have studied a comparative analysis on the interaction specificity of GPR17-ligands with hGPR17 and human purinergic G protein-coupled receptor (hP2Y1) receptors. Previously, we have simulated the interaction stability of synthetic ligands such as T0510.3657, AC1MLNKK, and MDL29951 with hGPR17 and hP2Y1 receptor in the lipid environment. In the present work, we have comparatively studied the protein-ligand interaction of hGPR17-T0510.3657 and P2Y1-MRS2500. Sequence analysis and structural superimposition of hGPR17 and hP2Y1 receptor revealed the similarities in the structural arrangement with the local backbone root mean square deviation (RMSD) value of 1.16 Å and global backbone RMSD value of 5.30 Å. The comparative receptor-ligand interaction analysis between hGPR17 and hP2Y1 receptor exposed the distinct binding sites in terms of geometrical properties. Further, the molecular docking of T0510.3657 with the hP2Y1 receptor have shown non-specific interaction. The experimental validation also revealed that Gi‐coupled activation of GPR17 by specific ligands leads to the adenylyl cyclase inhibition, while there is no inhibition upon hP2Y1 activation. Overall, the above findings suggest that T0510.3657-GPR17 binding specificity could be further explored for the treatment of numerous neuronal diseases. Communicated by Ramaswamy H. Sarma

Epitaxial nucleation, modulated structure of molecular aggregation, and enhanced thermal degradation temperature of poly(ethylene adipate): Effects of the naturally occurring uracil as a nucleator

AbstractWe fabricated a novel bio‐composite using the poly(ethylene adipate) (PEA) and naturally occurring uracil (URA), in which the URA with the full biocompatibility and degradability acted as a nucleator (NA). A distinct enhancement was found in the crystallizability, melt‐crystallization temperature (Tc), and crystallization rate of the PEA upon incorporation of the URA, an indicative of a good NA of the URA. The underlying nucleation mechanism of the PEA is the epitaxial nucleation, attributed to the extremely good matching between the crystal lattice of the PEA and URA. The H‐bond interaction exists between the carbonyl/ester segment of the PEA crystalline phase and URA. With loading of the URA, both the carbonyl and ester fell behind the CH2 in the segmental reorganization in the crystallization process of the PEA. The PEA/1%URA showed an enhancement in the Td in the TG measurement (i.e. increased stability in thermal degradation), mainly attributed to the flame retardance effect of the URA.

BNT162b2 Vaccine: Possible Codons Misreading, Errors in Protein Synthesis and Alternative Splicing's Anomalies

BNT162b2 vaccine against Covid-19 is composed of an RNA having 4284 nucleotides, divided into 6 sections, which bring the information to create a factory of S Spike proteins, the ones used by Sars-CoV-2 (Covid-19) to infect the host. After that, these proteins are directed outside the cell, triggering the immune reaction and antibody production. The problem is the heavy alteration of the mRNA: Uracil is replaced to fool the immune system with Ψ (Pseudouridine); the letters of all codon triplets are replaced by a C or a G, to extremely increase the speed of protein synthesis; replacement of some amino acids with Proline; addition of a sequence (3'-UTR) with unknown alteration. These impairments could cause strong doubts about the presence of codon usage errors. An eventual mistranslation has consequences on the pathophysiology of a variety of diseases. In addition, mRNA injected is a pre-mRNA, which can lead to the multiple mature mRNAs; these are alternative splicing anomalies, direct source of serious long-term harm on the human health. In essence, what will be created may not be identical with protein S Spike: just an error in translational decoding, codons misreading, production of different amino acids, then proteins, to cause serious long-term damage to human health, despite the DNA is not modified, being instead in the cell nucleus and not in the cytoplasm, where the modified mRNA arrives. However, in this case, the correlation between speed of synthesis and protein expression with synthesis errors, as well as the mechanism that could affect the translation of the sequence remain obscure, many trials have not yet been performed.

Structural Basis of SARS-CoV-2 Polymerase Inhibition by Favipiravir

SummaryThe outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into an unprecedented global pandemic. Nucleoside analogs, such as Remdesivir and Favipiravir, can serve as the first-line broad-spectrum antiviral drugs by targeting the viral polymerases. However, the underlying mechanisms for the antiviral efficacies of these drugs are far from well understood. Here, we reveal that Favipiravir, as a pyrazine derivative, could be incorporated into the viral RNA products by mimicking both adenine and guanine nucleotides. This drug thus inhibits viral replication mainly by inducing mutations in progeny RNAs, different from Remdesivir or other RNA-terminating nucleoside analogs that impair the elongation of RNA products. We further determined the cryo-EM structure of Favipiravir bound to the replicating polymerase complex of SARS-CoV-2 in the pre-catalytic state. This structure provides a missing snapshot for visualizing the catalysis dynamics of coronavirus polymerase, and reveals an unexpected base-pairing pattern between Favipiravir and pyrimidine residues that may explain its capacity for mimicking both adenine and guanine nucleotides. These findings shed light on the mechanism of coronavirus polymerase catalysis and provide a rational basis for developing antiviral drugs to combat the SARS-CoV-2 pandemic.

110th Anniversary: Selective Recognition of 5-Fluorouracil with Molecular Imprinting Membranes: Molecular Details

Molecular imprinting is a developing technique that provides materials with recognition sites having a predetermined selectivity for target molecules. The performance of the imprinted membranes is fundamentally influenced by hydrogen bonds between the template molecules and the polymeric matrices. In this work, molecular mechanics, quantum mechanics, and docking calculations were performed to understand the affinity of template vs monomers and the effect of porogenic solvents. The polymer system investigated methacrylic acid (MAA) imprinted with the 5- fluorouracil anticancer agent and with different analogues of 5-FU, i.e., uracil (UR), cytosine (CY), and 5-fluorocytosine (5-FC). The focus of the computational models was the analysis of the formation of the aggregates, composed of a specific template and monomers, based on their intermolecular interactions. The computational results confirmed the experimental selectivity of the material toward 5-FU demonstrating the important role of fluorine atoms altho...

Use of Cyano Probes in QM/MM Simulations to Study the Effect of Ion Concentration and Temperature of the Environment on a Uracil Nucleotide and DNA

Use of Cyano Probes in QM/MM Simulations to Study the Effect of Ion Concentration and Temperature of the Environment on a Uracil Nucleotide and DNA

Fingerprint Analysis and Multi-Component Determination of Ribonucleic Acid for Injection II Recipe by HPLC-DAD and LC-ESI-MS Methods.

Using high-performance liquid chromatography (HPLC), a quantitative method was developed to control the quality of ribonucleic acid for injection II (RA-II), which is used as an anti-cancer drug clinically in China. Using nuclease enzyme and under optimal hydrolysis conditions, the unstable RNA was hydrolyzed into stable nucleosides and nucleotides, which were easily detected by HPLC with diode array detection. Furthermore, by analyzing HPLC chromatograms of 10 batches of samples, six common peaks, namely, cytosine nucleotide, uracil nucleotide, adenine nucleotide, guanine nucleotide, guanosine and adenine, were identified by a HLPC coupled with electrospray ionization mass spectrometry. The similarity, the relative retention time and the relative peak area of each common peak, relative to the reference peak, were calculated to obtain the HPLC fingerprints, and their values were within the scope of the provisions. More importantly, all six components were simultaneously determined. Overall, the developed method in this investigation proved to be a useful tool for monitoring the quality of RA-II in terms of their batch variations and the raw material sources.

GPR17 mediates ischemia-like neuronal injury via microglial activation.

GPR17 is a G (i)-coupled dual receptor, linked to P2Y and CysLT receptors stimulated by uracil nucleotides and cysteinyl leukotrienes, respectively. Recent evidence has demonstrated that GPR17 inhibition ameliorates the progression of cerebral ischemic injury by regulating neuronal death and microglial activation. The present study aimed to assess the detailed regulatory roles of this receptor in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemia-like injury in vitro and explore the underlying mechanism. The results demonstrated that OGD/R induced ischemic neuronal injury and microglial activation, including enhanced phagocytosis and increased inflammatory cytokine release in neuron‑glial mixed cultures of cortical cells. GPR17 upregulation during OGD/R was spatially and temporally correlated with neuronal injury and microglial activation. In addition, GPR17 knockdown inhibited OGD/R-induced responses in neuron-glial mixed cultures. GPR17 knockdown also attenuated cell injury induced by the agonist leukotriene D4 (LTD4) or uridine 5′-diphosphate (UDP) in neuron-glial mixed cultures. However, GPR17 knockdown did not affect OGD/R-induced ischemic neuronal injury in primary cultures of neurons. In primary astrocyte cultures, neither GPR17 nor OGD/R induced injury. By contrast, GPR17 knockdown ameliorated OGD/R-induced microglial activation, boosting phagocytosis and inflammatory cytokine release in primary microglia cultures. Finally, the results demonstrated that the conditioned medium of microglia pretreated with OGD/R induced neuronal death, and the neuronal injury was significantly inhibited by GPR17 knockdown. These findings suggested that GPR17 may mediate ischemia-like neuronal injury and microglial activation in vitro; however, the protective effects on ischemic neuronal injury might depend upon microglial activation. Whether GPR17 regulates neuronal injury mediated by oligodendrocyte linkage remains to be investigated.

P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDP-glucose (< 30 μM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 μM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.