Atherosclerosis is induced by lipid accumulation, inflammation, and endothelial dysfunction, and is the leading cause of death from cardiovascular disease worldwide. The P2Y6 receptor can be activated by the extracellular release of UDP. The evidence from the last decade has highlighted its critical therapeutic effect in atherosclerosis, yet with unclear mechanisms. This review introduced the P2Y6 receptor in atherosclerosis, and its mechanisms of atherosclerosis-promoting in macrophages, endothelial cells, and vascular smooth muscle cells. Finally, we discussed the development and potential of P2Y6 receptor antagonists in treating atherosclerosis.

Despite the notable success of cancer immunotherapy, its effectiveness is often limited in a significant proportion of patients, highlighting the need to explore alternative tumor immune evasion mechanisms. Adenosine, a key metabolite accumulating in hypoxic tumor regions, has emerged as a promising target in oncology. Inhibiting the adenosinergic pathway not only inhibits tumor progression but also holds potential to enhance immunotherapy outcomes. Multiple therapeutic strategies targeting this pathway are being explored, ranging from preclinical studies to clinical trials. This review examines the complex interactions between adenosine, its receptors, and the tumor microenvironment, proposing strategies to target the adenosinergic axis to boost anti-tumor immunity. It also evaluates early clinical data on pharmacological inhibitors of the adenosinergic pathway and discusses future directions for improving clinical responses.

The activation of brown adipose tissue (BAT) or the browning of white adipose tissue (WAT) represents a promising therapeutic strategy for obesity. Arctiin (ARC), a lignan compound known for its anti-inflammatory, anti-tumor, and hypoglycemic properties, has not been fully elucidated regarding its effects and mechanisms on obesity.

In the present study, we established both high-fat diet-induced obese mouse models and mature adipocyte cultures to comprehensively investigate the therapeutic effects of ARC on obesity. Systemic energy metabolism and thermogenic capacity were assessed through metabolic cage monitoring and cold stimulation tests. Histopathological alterations in adipose tissues were examined using hematoxylin and eosin (H&E) staining, while key gene expression in adipocytes was determined by Western blotting (WB), immunohistochemistry, and immunofluorescence staining. To further elucidate the molecular mechanisms underlying ARC's anti-obesity effects, we employed an integrated approach combining network pharmacology analysis, molecular docking simulations, cellular thermal shift assay (CETSA), and WB to identify potential molecular targets and delineate the associated signaling pathways modulated by ARC treatment.

In diet-induced obese mice, ARC administration at doses of 20 and 60 mg/kg/day ameliorated metabolic dysfunction through enhanced WAT browning and increased energy expenditure. In C3H10T1/2-induced adipocytes, ARC upregulated the protein expression of uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and other brown-specific marker genes, promoting mitochondrial function and browning of adipocytes. Mechanistically, our findings suggest that ARC may promote adipocyte browning via the A2AR-cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway.

In summary, ARC exerts protective effects against obesity by promoting the browning of white adipocytes and holds promise as a potentially beneficial therapeutic agent for the treatment of obesity.

For steady transgenic expression, lentiviral vector-mediated gene delivery is a commonly used technique. One question that needs to be explored is how external lentiviral vectors and overexpressed genes perturb cellular homeostasis, potentially altering transcriptional networks. In this study, two Human Embryonic Kidney 293T (HEK293T)-derived cell lines were established via lentiviral transduction, one overexpressing green fluorescent protein (GFP) and the other co-overexpressing GFP and ADORA3 following puromycin selection to ensure stable genomic integration. Genes with differentially transcript utilization (gDTUs) and differentially expressed genes (DEGs) across cell lines were identified after short-read and long-read RNA-seq. Only 31 genes were discovered to have changed in expression when GFP was expressed, although hundreds of genes showed variations in transcript use. In contrast, even when co-overexpression of GFP and ADORA3 alters the expression of more than 1000 genes, there are still less than 1000 gDTUs. Moreover, DEGs linked to ADORA3 overexpression play a major role in RNA splicing, whereas gDTUs are highly linked to a number of malignancies and the molecular mechanisms that underlie them. For the analysis of gene expression data from stable cell lines derived from HEK293T, our findings provide important insights into changes in gene expression and alternative splicing.

Tanycytes are a pivotal component of the hypothalamic network that controls energy homeostasis. Despite their importance, the regulatory mechanisms governing tanycyte-neuron interactions in response to metabolic signals remain unexplored. Here we report that adenosine signaling between tanycytes and AGRP/NPY neurons is crucial for tanycytic metabolic regulation mediated by translocator protein 18 kDa (TSPO). Tanycyte-specific Tspo-knockout mice displayed reduced food consumption and weight loss associated with the downregulation of Agrp and Npy expression under high-fat diet feeding. Tspo-deficient tanycytes had elevated levels of intracellular ATP, which was released via connexin 43 hemichannels and extracellularly converted into adenosine by tanycytic ectonucleotidases. The adenosine signal was perceived by adenosine A1 receptors on adjacent AGRP/NPY neurons, reducing ERK phosphorylation, which in turn downregulated Agrp and Npy expression. Our findings underscore the anorexic role of adenosine as a gliotransmitter in the intricate communication between tanycytes and neurons for regulating appetite and body weight.

Pulsed Electromagnetic Field (PEMF) therapy has been shown to have substantial suppressive effects on inflammation and is a promising treatment for the modulation of inflammation. Several in vitro and in vivo studies have shown that PEMFs profoundly suppress inflammatory pathways, such as the NF-κB and MAPK signaling pathways, by lowering cytokine levels and improving extracellular matrix synthesis. This review describes studies, ranging from in vitro to clinical, that investigate the lesser-known roles of PEMF in the modulation of inflammation in soft tissue wound, cartilage, and joint healing, alongside angiogenesis. Mechanistically, PEMFs act via adenosine receptors, specifically A2A, which play a key role in inflammation modulation and tissue repair. In some clinical trials, PEMF has yielded short-term symptom relief and functional improvements in early-stage osteoarthritis patients, arthroscopy patients, and anterior cruciate ligament reconstruction patients.

Multiple Sclerosis (MS) is an immune-mediated disease with miscellaneous etiological origins. Given caffeine's neuroprotective and anti-inflammatory attributes and its potential influence on MS risk, and to address the conflict in the clinical evidence, this study aims to comprehensively review the existing literature on the association between coffee consumption and the risk of MS.

Following the PRISMA 2020 guidelines, a systematic search in PubMed, Scopus, Web of Science, and Embase for the studies published up to January 2024 was conducted. Studies that assessed the relationship between coffee intake and the risk of MS were included, and reviews, case reports, non-English papers, in vitro and animal studies, and conference abstracts were excluded. The risk of bias was assessed using the JBI checklists, and meta-analyses were conducted based on odds ratio (OR) using the fourth version of CMA software.

Out of 604 initial records, 10 observational studies with 19,430 participants met the inclusion criteria. The included case-control studies showed an overall high quality. Meta-analysis revealed a reduction in MS development in coffee consumers both before (OR: 0.66; 95 % CI: 0.49-0.90; p-value: 0.008; I2: 89.65 %; p-value for heterogeneity<0.001) and after adjustment for possible confounders (adjusted OR: 0.42; 95 % CI: 0.20-0.90; p-value: 0.025; I2: 89.65 l; p-value for heterogeneity<0.001).

Coffee consumption, may decrease the risk of MS; however, further well-designed prospective studies are required to ascertain this association. PROSPERO registration number: CRD42023484298.

The gastrointestinal tract is highly sensitive to ionizing radiation (IR), which causes radiation-induced intestinal injury (RIII). There are no effective drugs available for RIII in routine clinical treatment, which is a major limiting factor during the process of radiotherapy for pelvic abdominal malignancies. In this study, we aimed to elucidate the potential of probiotic Bacillus coagulans BC99 (B.coagulans BC99) in preventing RIII. C57BL/6J mice were gavage-administered with B.coagulans BC99 for 30 days and then exposed to a single dose of 12 Gy x-ray whole abdominal irradiation (WAI). B.coagulans BC99 treatment could mitigate RIII by preventing weight loss, maintaining the integrity of intestinal structure and barrier, improving inflammatory symptoms, modulating oxidative stress, and regulating the composition of gut microbiota, thereby reestablishing intestinal homeostasis. In addition, the potential radioprotective mechanism of B.coagulans BC99 was closely related to the gut microbiota-derived metabolites. This study offers a novel perspective for advancing probiotic-based treatments for RIII and enhancing strategies for the prevention of RIII.

Bone fractures and related complications are a significant concern for older adults, particularly with the growing aging population. Therapeutic interventions that promote bone tissue regeneration are attractive for geriatric fracture repair. Extracellular adenosine plays a key role in bone homeostasis and regeneration. Herein, we examined the changes in extracellular adenosine with aging and the potential of local delivery of adenosine to promote fracture healing using aged mice. Extracellular adenosine level was found to be significantly lower in aged bone tissue compared to young mice. Concomitantly, the ecto-5'-nucleotidase CD73 expression was also lower in aged bone. Local delivery of adenosine using injectable, in situ curing microgel delivery units yielded a pro-regenerative environment and promoted fracture healing in aged mice. This study offers new insights into age-related physiological changes in adenosine levels and demonstrates the therapeutic potential of adenosine supplementation to circumvent the compromised healing of geriatric fractures.

G protein-coupled receptors (GPCRs) are the largest human membrane protein family that transduce extracellular signals into cellular responses. They are major pharmacological targets, with approximately 26% of marketed drugs targeting GPCRs, primarily at their orthosteric binding site. Despite their prominence, predicting the pharmacological effects of novel GPCR-targeting drugs remains challenging due to the complex functional dynamics of these receptors. Recent advances in X-ray crystallography, cryo-electron microscopy, spectroscopic techniques and molecular simulations have enhanced our understanding of receptor conformational dynamics and ligand interactions with GPCRs. These developments have revealed novel ligand-binding modes, mechanisms of action and druggable pockets. In this Review, we highlight such aspects for recently discovered small-molecule drugs and drug candidates targeting GPCRs, focusing on three categories: allosteric modulators, biased ligands, and bivalent and bitopic compounds. Although studies so far have largely been retrospective, integrating structural data on ligand-induced receptor functional dynamics into the drug discovery pipeline has the potential to guide the identification of drug candidates with specific abilities to modulate GPCR interactions with intracellular effector proteins such as G proteins and β-arrestins, enabling more tailored selectivity and efficacy profiles.

The interplay between Parkinson's disease (PD) and sleep disturbances suggests that sleep problems constitute a risk factor for PD progression, but the underlying mechanisms remain unclear. Microglial activation and oxidative stress are considered to play an important role in the pathogenesis of aging and neurodegenerative diseases. We hypothesized that sleep deprivation (SD) could exacerbate PD progression via modulating microglial activation and oxidative stress. To test this hypothesis, we established a PD mouse model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), then subjected the mice to SD. A battery of behavioral tests, including rotarod, pole, adhesive removal, and open field tests, were used to assess motor function. Our study showed that SD exacerbated motor deficits, loss of tyrosine hydroxylase (TH), microglial activation and oxidative stress damage in PD model mice. Fecal microbiota transplantation experiments revealed that SD mediated PD progression, microglial activation and oxidative stress via the gut microbiota. 16S rRNA sequencing analysis indicated that SD increased the abundances of bacteria such as Bacteroidaceae, while decreasing the abundances of bacteria including Lactobacillus. Non-targeted metabolomic analysis of gut microbiota-derived metabolites revealed that SD significantly increased the production of adenosine (ADO), a purine metabolite. Probiotic supplementation reversed the effects of SD on motor deficits, dopaminergic neuron loss, microglial activation and oxidative stress damage in PD mice; it also decreased SD-induced ADO production. Administration of Adenosine A2A receptor (A2AR) inhibitors, Istradefylline (Ist), attenuated the roles of SD and ADO in promoting microglial activation, oxidative stress and PD progression. Taken together, our findings indicate that SD accelerates PD progression via regulating microbiota associated microglial activation and oxidative stress, suggesting that efforts to improve sleep quality can be used to prevent and treat PD.

The Adenosine A2B receptor (A2BAR) is considered a novel potential target for the immunotherapy of cancer, and A2BAR antagonists have an inhibitory effect on tumor growth, proliferation, and metastasis. In our previous studies, we identified a class of benzimidazole-pyrazine scaffolds whose derivatives exhibited the antagonistic effect but lacked subtype selectivity towards A2BAR. In this work, we developed a scaffold-based protocol that incorporates a deep generative model and multilayer virtual screening to design benzimidazole-pyrazine derivatives as potential selective A2BAR antagonists. By utilizing a generative model with reported A2BAR antagonists as the training set, we built up a scaffold-focused library of benzimidazole-pyrazine derivatives and processed a virtual screening protocol to discover potential A2BAR antagonists. Finally, five molecules with different Bemis-Murcko scaffolds were identified and exhibited higher binding free energies than the reference molecule 12o. Further computational analysis revealed that the 3-benzyl derivative ABA-1266 presented high selectivity toward A2BAR and showed preferred draggability, providing future potent development of selective A2BAR antagonists.

The previous work discovers the potential of adenosine monophosphate (AMP) to alleviate obesity-related metabolic diseases, but the underlying molecular mechanisms remain incompletely understood. Here, AMP is confirmed to enhance white fat decomposition and improve abnormal glucose and lipid metabolism in mice fed with a high-fat (HF) diet. Mechanically, AMP is converted to adenosine (ADO) through ecto-5'-nucleotidase (CD73), and adenosine A2A receptor (ADORA2A) signaling activation is involved in the down-regulation of methylation in white adipose tissue, thereby reducing the hormone-sensitive lipase (HSL) methylation level and promoting HSL transcription and white fat decomposition. Moreover, the metabolic benefits of AMP are found to be partially eliminated in ADORA2A knockout mice, but re-expression of ADORA2A can reproduce the AMP-induced metabolic regulation in white fat. These findings reveal the mechanism that AMP, as the upstream of ADO, stimulates ADORA2A signaling and white fat DNA methylation to participate in the anti-obesity effect.

Over the past decade, mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) have emerged as promising therapeutics, shifting the focus from MSC engraftment or differentiation to their secretion of sEVs-particularly those under 200 nm-that mediate regenerative and immunomodulatory functions. Transitioning from cell therapies to sEV-based therapies offers clinical advantages, including reduced challenges with cell viability, storage, and administration, and improved pharmacological predictability. However, manufacturing MSC-sEV products faces challenges in defining critical quality attributes (CQAs) for consistent identity and potency. Variability arises from differences in cell sources, culture conditions, enrichment techniques, and the inherent heterogeneity of MSCs. Even the use of immortalized clonal MSC lines may not fully eliminate variability, as factors such as developmental processes, epigenetic modifications, or genetic drift could lead to the re-emergence of heterogeneity. Establishing robust potency CQAs is further complicated by the complex, multimodal modes of action of MSC-sEV products, which involve diverse mechanisms impacting various cell types and processes. Traditional models of EV mediated signalling suggesting direct internalization of sEVs by target cells are increasingly challenged due to inefficient EV-uptake and the high therapeutic efficacy observed. Instead, the Extracellular Modulation of Cells by EVs (EMCEV) model proposes that MSC-sEVs exert their effects by modulating the extracellular environment, enabling a "one EV to many cells" interaction. In conclusion, while MSC-sEV products hold significant therapeutic promise due to their multimodal action and functional redundancy, manufacturing challenges and the complexity of defining potency CQAs remain hurdles to clinical translation. A pragmatic approach focusing on identifying key potency-related CQAs based on specific mechanisms of action-while recognizing that "the process defines the product"-may facilitate the advancement of MSC-sEV therapeutics into clinical applications.

Glial-origin brain tumors, particularly glioblastomas (GBMs), are known for their devastating prognosis and are characterized by rapid progression and fatal outcomes. Despite advances in surgical resection, complete removal of the tumor remains unattainable, with residual cells driving recurrence that is resistant to conventional therapies. The GBM tumor microenviroment (TME) significantly impacts tumor progression and treatment response. In this review, we explore the emerging role of purinergic signaling, especially the P2X7 receptor (P2X7R). Due to its unique characteristics, it plays a key role in tumor progression and offers a potential therapeutic strategy for GBM through TME modulation. We discuss also the emerging role of the P2X4 receptor (P2X4R) as a promising therapeutic target. Overall, targeting purinergic signaling offers a potential approach to overcoming current GBM treatment limitations.

Metabolic dysfunction-associated steatotic liver (MASLD) progression is driven by chronic inflammation and fibrosis, largely influenced by Kupffer cell (KC) dynamics, particularly replenishment of pro-inflammatory monocyte-derived KCs (MoKCs) due to increased death of embryo-derived KCs. Adenosine A3 receptor (A3AR) plays a key role in regulating metabolism and immune responses, making it a promising therapeutic target. This study aimed to investigate the impact of selective A3AR antagonism for regulation of replenished MoKCs, thereby improving MASLD.

A3AR expression was significantly elevated in KCs from both patients with MASLD and fast-food diet (FFD)-fed mice. A3AR knockout (KO) mice displayed marked improvements in hepatic inflammation and fibrosis along with a reduction in CLEC4F-positive KCs. The spatial transcriptomics of these KCs revealed disrupted mitochondrial integrity, increased oxidative stress, and enhanced cell death due to A3AR deletion. Similarly, in vivo FM101 treatment, a highly potent and selective antagonist of A3AR with a truncated 4'-thioadenosine structure, mitigated FFD-induced MASLD in mice. Mechanistically, FM101 induces β-arrestin2-mediated A3AR degradation, leading to mitochondrial dysfunction-mediated necroptosis in KCs. Consistently, A3AR was highly expressed in monocyte-derived macrophages in MASLD patients, with strong correlations with macrophage activation and monocyte chemoattractant gene sets. Thus, FM101 induced necroptosis in pro-inflammatory MoKCs, facilitating anti-inflammatory effects.

This study demonstrated that inhibiting A3AR via FM101 or genetic deletion alleviates MASLD by inducing mitochondrial dysfunction and subsequent necroptosis in MoKCs, establishing FM101 as a promising therapeutic strategy for MASLD.

Adenosine serves as a critical homeostatic regulator, exerting influence over physiological and pathological conditions in the cardiovascular system. During cellular stress, increased extracellular adenosine levels have been implicated in conferring cardioprotective effects through the activation of adenosine receptors with the A1 adenosine receptor subtype showing the highest expression in the heart. A1 adenosine receptor stimulation inhibits adenylyl cyclase activity via heterotrimeric Gi proteins, leading to the activation of distinct downstream effectors involved in cardiovascular homeostasis. While the comprehensive characterization of the pharmacological functions and intracellular signaling pathways associated with the A1 adenosine receptor subtype is still ongoing, this receptor is widely recognized as a crucial pharmacological target for the treatment of various states of cardiovascular diseases (CVDs). In this review, we focus on elucidating signal transduction of A1 adenosine receptor, particularly Gi protein-dependent and -independent pathways, and their relevance to cardiovascular protective effects as well as pathological consequences during cellular and tissue stresses in the cardiovascular system. Additionally, we provide comprehensive updates and detailed insights into a range of A1 adenosine receptor agonists and antagonists, detailing their development and evaluation through preclinical and clinical studies with a specific focus on their potential for the management of CVDs, especially heart diseases.

Immune checkpoint inhibitors are effective treatments for HCC; however, their therapeutic efficacy is often limited by the development of drug resistance. Therefore, investigating new combination therapeutics involving immune checkpoint inhibitors is critical to improving patient prognosis. In this study, we investigated the therapeutic effect of cordycepin (COR) in HCC and its synergistic effect with anti-programmed cell death ligand 1 (anti-PD-L1) immunotherapy.

We selected 2 HCC cell lines to investigate the effects of COR on HCC growth using in vivo and in vitro experiments. We performed RNA sequencing of the MHCC97H cell line treated with or without COR to understand the underlying mechanism and identify the key regulatory genes. Through in vivo and in vitro experiments on gene knockdown cells, we identified thioredoxin-interacting protein as a key molecule involved in the role of COR. Next, we used mouse subcutaneous and orthotopic tumor models to evaluate the therapeutic effects of COR, atezolizumab (a programmed death-ligand 1 [PD-L1] inhibitor), or their combination. Multiple immunofluorescence staining revealed that the combination of atezolizumab and COR therapy greatly increased the number of tumor-infiltrating CD8+ T cells and PD-L1 expression in HCC compared to monotherapy.

Our study revealed that COR significantly inhibited HCC growth both in vitro and in vivo. Mechanistically, we showed that COR induces endoplasmic reticulum stress, which upregulates thioredoxin-interacting protein expression and leads to HCC cell pyroptosis. In addition, the combination treatment with COR and PD-L1 inhibitors profoundly inhibited HCC.

Overall, our study successfully established a combined therapeutic strategy using COR and PD-L1 inhibitors. This strategy has significant synergistic effects on cancer cells, highlighting its importance in cancer therapy.

Purinergic P2 receptors, which can be divided into ionotropic P2X receptors and metabotropic P2Y receptors, mediate cellular signal transduction of purine or pyrimidine nucleoside triphosphates and diphosphate. Based on the wide expression of purinergic P2 receptors in tissues and organs, their significance in homeostatic maintenance, metabolism, nociceptive transmission, and other physiological processes is becoming increasingly evident, suggesting that targeting purinergic P2 receptors to regulate biological functions and signal transmission holds significant promise for disease treatment.

This review highlights the detailed mechanisms by which purinergic P2 receptors engage in physiological and pathological progress, as well as providing prospective strategies for discovering clinical drug candidates.

The purinergic P2 receptors regulate complex signaling and molecular mechanisms in nervous system, digestive system, immune system and as a result, controlling physical health states and disease progression. There has been a significant rise in research and development focused on purinergic P2 receptors, contributing to an increased number of drug candidates in clinical trials. A few influential pioneers have laid the foundation for advancements in the evaluation, development, and of novel purinergic P2 receptors modulators, including agonists, antagonists, pharmaceutical compositions and combination strategies, despite the different scaffolds of these drug candidates. These advancements hold great potential for improving therapeutic outcomes by specifically targeting purinergic P2 receptors.

G protein-coupled receptors (GPCRs) can transmit signals via G protein-dependent or independent pathways due to the conformational changes of receptors and ligands, which is called biased signaling. This concept posits that ligands can selectively activate a specific signaling pathway after receptor activation, facilitating downstream signaling along a preferred pathway. Biased agonism enables the development of ligands that prioritize therapeutic signaling pathways while mitigating on-target undesired effects. As a class of GPCRs located on the surface of cell membranes, the discovery and clinical implementation of adenosine and P2Y receptors purinergic signaling modulators have progressed dramatically. However, many preclinical drug candidates targeting purinergic receptors have failed in clinical trials due to limited efficacy and/or severe on-target undesired effects. To overcome the key barriers typically encountered when transitioning ligands into the clinic, the renewed impetus has focused on the modulation of purinergic receptor function by exogenous agonists/antagonists and allosteric modulators to exploit biased agonism. This article provides a brief overview of the research progress on the mechanism of purinergic biased signal transduction from the conformational changes of purinergic GPCRs and biased ligands primarily, and highlights therapeutically relevant biased agonism at purinergic receptors.

Microvascular ischemia, especially in the heart and kidneys, is associated with inflammation and metabolic perturbation, resulting in cellular dysfunction and end-organ failure. Heightened production of adenosine from extracellular nucleotides released in response to inflammation results in protective effects, inclusive of adaptations to hypoxia, endothelial cell nitric oxide release with the regulation of vascular tone, and inhibition of platelet aggregation. Purinergic signaling is modulated by ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39, which is the dominant factor dictating vascular metabolism of extracellular ATP to adenosine throughout the cardiovascular tissues. Excess levels of extracellular purine metabolites, however, have been associated with metabolic and cardiovascular diseases. Physiological estrogen signaling is anti-inflammatory with vascular protective effects, but pharmacological replacement use in transgender and postmenopausal individuals is associated with thrombosis and other side effects. Crucially, the loss of this important sex hormone following menopause or with gender reassignment is associated with worsened pro-inflammatory states linked to increased oxidative stress, myocardial fibrosis, and, ultimately, diastolic dysfunction, also known as Yentl syndrome. While there is a growing body of knowledge on distinctive purinergic or estrogen signaling and endothelial health, much less is known about the relationships between the two signaling pathways. Continued studies of the interactions between these pathways will allow further insight into future therapeutic targets to improve the cardiovascular health of aging women without imparting deleterious side effects.

Inhibition of the adenosine 2A receptor (A2AR) is recognized as a promising immunotherapeutic strategy but is challenged by the ubiquity of A2AR function in the immune system. To develop a safe yet efficacious immunotherapy, the discovery of a novel negative allosteric modulator (NAM) was preferred. Leveraging an in-house, sensitive, high-throughput screening cellular assay, novel A2AR NAM scaffolds were identified, followed by an extensive structure-activity relationship (SAR) study, leading to the discovery of potent 2-amino-3,5-dicyanopyridine derivatives. The allosteric mode of action of active compounds was confirmed by progressive fold-shift assay, nonlinearity of the Schild plot analysis, biophysical measurements, and retained satisfactory potencies in high-adenosine concentrations. Further correlation of A2AR engagement and downstream signaling was done in a human blood translational assay, clearly showcasing the potential of A2AR allosteric modulation as a novel approach for efficient and safer cancer immunotherapies.

Purinergic signaling pathways play crucial roles in regulating hemostatic and inflammatory responses, both of which are impacted by scorpion envenomation. Scorpion venoms are complex mixtures of various toxins, such as peptides, enzymes, and nucleotides. Previous research showed that the action of scorpion toxins on the purinergic system stems from their effects on purinergic receptors. Additionally, a study identified a putative ectonucleotidase in scorpion venom. This study aimed to investigate the ability of Tityus confluens venom (10, 50, and 100 µg/mL) to metabolize adenine nucleotides and its potential effects on purinergic enzyme activity in rat platelets and lymphocytes. The effects of T. confluens venom on E-NTPDase (ATP and ADP hydrolysis), E-5'-NT (AMP hydrolysis), and E-ADA (ADO hydrolysis) activities were analyzed. The results revealed that crude venom from T. confluens exhibited ATP hydrolysis activity at all tested concentrations. In lymphocytes, ADP hydrolysis was inhibited by 100 µg/mL crude venom, whereas ADO hydrolysis was increased by all venom concentrations. In platelets, ATP hydrolysis was inhibited by 50 and 100 µg/mL crude venom, whereas AMP and ADO hydrolysis were inhibited by all concentrations. When considered collectively, the data suggested an elevation in extracellular ATP levels and a reduction in extracellular ADO. These findings are in alignment with clinical manifestations of scorpion envenomation characterized by a pro-inflammatory milieu. Furthermore, this study demonstrated the intrinsic ATPase activity of T. confluens venom and its ability to modulate E-NTPDase, E-5'-NT, and E-ADA activities in rat blood cells.

Decidual macrophages (dMφ) are pivotal in maintaining maternal-fetal immune tolerance during normal pregnancy by expressing a range of immune-suppressive molecules, including CD73. It has been demonstrated that Toxoplasma gondii (T. gondii) infection during pregnancy can impair dMφ function, potentially leading to adverse pregnancy outcomes, through downregulation of these inhibitory molecules. T. gondii rhoptry protein 18 (TgROP18), a key virulence factor of T. gondii, is associated with the incapacitation of the host's innate and adaptive immune responses to protect the parasite from elimination. However, the role of TgROP18 in modulating CD73 expression on dMφ and the underlying mechanisms remain to be elucidated.

Wild-type (WT) and CD73-deficient (CD73-/-) pregnant mice were subjected to intraperitoneal injection of T. gondii RH or RH-Δrop18 on gestational day (Gd) 8, and subsequently euthanized on Gd 14. Pregnancy outcomes were then evaluated, and the expression levels of CD73, arginase 1 (Arg-1), and interleukin 10 (IL-10) were quantified by flow cytometry. Mononuclear cells isolated from the human aborted decidual tissues were also infected with T. gondii RH or RH-Δrop18 for the analysis of CD73 expression with flow cytometry. Additionally, infected human dMφ were used to assess the expression levels of CD73, Arg-1, IL-10, and their associated signaling molecules by western blot analysis. Furthermore, chromatin immunoprecipitation (ChIP) assays were performed to validate the involved signaling pathways.

Compared with the T. gondii RH-infected group, milder adverse pregnancy outcomes and attenuated expression levels of CD73 on dMφ were observed in T. gondii RH-Δrop18-infected pregnant mice and human decidual tissues. Lysine-specific histone demethylase1 (LSD1) and snail family transcriptional repressor 1 (SNAIL1) were found to be involved in the downregulation of CD73 expression on dMφ following T. gondii infection. Subsequently, reduced expression of CD73 contribute to the downregulation of Arg-1 and IL-10 expression through adenosine A2a receptor (A2AR) / protein kinase A (PKA) / phosphorylated cAMP-response element binding protein (p-CREB) / CCAAT enhancer binding protein B (C/EBPβ) pathway.

TgROP18 can significantly reduce CD73 expression on dMφ through LSD1/SNAIL1 pathway, subsequently leading to the decreased expression levels of Arg-1 and IL-10 via adenosine/A2AR/PKA/p-CREB/C/EBPβ pathway, which ultimately contributes to maternal-fetal tolerance dysfunction of dMφ.

Body mass index (BMI) is considered an important factor in tumor prognosis, but its role in gastric cancer (GC) remains controversial. There is a lack of studies exploring the effect of BMI on gastric cancer from the perspective of intratumoral microbiota. This study aimed to compare and analyze the differences in and functions of intratumoral microbiota among GC patients with varying BMIs, aiming to ascertain whether specific microbial features are associated with prognosis in low-BMI (LBMI) gastric cancer patients.

A retrospective analysis of the clinicopathological features and prognosis of 5567 patients with different BMIs was performed between January 2010 and December 2019. Tumor tissues from 189 GC patients were collected for 16S rRNA sequencing, 64 samples were selected for transcriptome sequencing, and 57 samples were selected for untargeted metabolomic analysis.

Clinical cohort analysis revealed that GC patients with a low BMI presented poorer clinical and pathological characteristics than those with a non-low-BMI (NLBMI). LBMI was identified as a significant independent risk factor for adverse prognosis, potentially exerting immunosuppressive effects on postoperative adjuvant chemotherapy. 16S rRNA sequencing revealed no significant differences in the alpha and beta diversity of the intratumoral microbiota between the two groups of GC patients. However, LEfSe analysis revealed 32 differential intratumoral microbiota between the LBMI and NLBMI groups. Notably, the genus Abiotrophia was significantly enriched in the LBMI group. Further in-depth analysis indicated that the genus Abiotrophia was inversely associated with eosinophils, P2RY12, and SCN4B genes, and positively linked with LGR6 in LBMI gastric cancer patients. Metabolomic assessments revealed that LBMI was positively associated with purine metabolites, specifically guanine and inosine diphosphate (IDP).

In conclusion, LBMI is an independent risk factor for poor prognosis in gastric cancer patients and may have an inhibitory effect on postoperative adjuvant chemotherapy. Intratumor flora of gastric cancer patients with different BMI levels differed, with different immune cell infiltration and metabolic characteristics. The genus Abiotrophia may promote gastric cancer development and progression by regulating eosinophils and the purine metabolism pathway, which provides a new idea for the precise treatment of gastric cancer.

This review explores the essential roles of purine metabolism including the catabolic product, uric acid, in the development of dopaminergic neurons of the substantia nigra pars compacta. The high energy requirements of the substantia nigra pars compacta alongside necessary purinergic neurotransmission and the influence of oxidative stress during development makes these neurons uniquely susceptible to changes in purine metabolism. Uric acid's role as a central nervous system antioxidant may help to ameliorate these effects in utero. Understanding the mechanisms by which purines and uric acid influence development of the substantia nigra pars compacta can help further explain neurologic consequences of inborn errors of purine metabolism, such as Lesch-Nyhan disease.

Cantharidin and sodium fluoride inhibit the activity of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) and increase the force of contraction in human atrial preparations. R-phenylisopropyl adenosine (R-PIA) acts as an agonist at A1-adenosine receptors. R-PIA exerts a negative inotropic effect on human atria. The effect of R-PIA-and its various manifestations-are currently explained as a function of the inhibition of sarcolemmal adenylyl cyclase activity and/or opening of sarcolemmal potassium channels. We hypothesise that cantharidin and sodium fluoride may attenuate the negative inotropic effect of R-PIA. During open heart surgery, trabeculae carneae from the right atrium were obtained for human atrial preparations (HAPs). These trabeculae were mounted in organ baths and electrically stimulated at 1 Hz. Furthermore, we studied isolated electrically stimulated left atrial (LA) preparations from female wild-type mice (CD1). The force of contraction was recorded under isometric conditions. R-PIA (1 µM) exerted a rapid negative inotropic effect in the HAPs and mice LA preparations. These negative inotropic effects of R-PIA were attenuated by pre-incubation for 30 min with 100-µM cantharidin in HAPs, but not in mice LA preparations. Adenosine signals via A1 receptors in a species-specific pathway in mammalian atria. We postulate that R-PIA, at least in part, exerts a negative inotropic effect via activation of serine/threonine phosphatases in the human atrium.

Brain plasticity is the ability of the nervous system to change its structure and functioning in response to experiences. These changes occur mainly at synaptic connections, and this plasticity is named synaptic plasticity. During postnatal development, environmental influences trigger changes in synaptic plasticity that will play a crucial role in the formation and refinement of brain circuits and their functions in adulthood. One of the greatest challenges of present neuroscience is to try to explain how synaptic connections change and cortical maps are formed and modified to generate the most suitable adaptive behavior after different external stimuli. Adenosine is emerging as a key player in these plastic changes at different brain areas. Here, we review the current knowledge of the mechanisms responsible for the induction and duration of synaptic plasticity at different postnatal brain development stages in which adenosine, probably released by astrocytes, directly participates in the induction of long-term synaptic plasticity and in the control of the duration of plasticity windows at different cortical synapses. In addition, we comment on the role of the different adenosine receptors in brain diseases and on the potential therapeutic effects of acting via adenosine receptors.

Patients with hemorrhagic stroke have high rates of morbidity and mortality, and drugs for prevention are very limited. Mendelian randomization (MR) analysis can increase the success rate of drug development by providing genetic evidence. Previous MR analyses only analyzed the role of individual drug target genes in hemorrhagic stroke; therefore, we used MR analysis to systematically explore the druggable genes for hemorrhagic stroke. We sequentially performed summary-data-based MR analysis and two-sample MR analysis to assess the associations of all genes within the database with intracranial aneurysm, intracerebral hemorrhage, and their subtypes. Validated genes were further analyzed by colocalization. Only genes that were positive in all three analyses and were druggable were considered desirable genes. We also explored the mediators of genes affecting hemorrhagic stroke incidence. Finally, the associations of druggable genes with other cardiovascular diseases were analyzed to assess potential side effects. We identified 56 genes that significantly affected hemorrhagic stroke incidence. Moreover, TNFSF12, SLC22A4, SPARC, KL, RELT, and ADORA3 were found to be druggable. The inhibition of TNFSF12, SLC22A4, and SPARC can reduce the risk of intracranial aneurysm, subarachnoid hemorrhage, and intracerebral hemorrhage. Gene-induced hypertension may be a potential mechanism by which these genes cause hemorrhagic stroke. We also found that blocking these genes may cause side effects, such as ischemic stroke and its subtypes. Our study revealed that six druggable genes were associated with hemorrhagic stroke, and the inhibition of TNFSF12, SLC22A4, and SPARC had preventive effects against hemorrhagic strokes.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with mild cognitive impairment (MCI) often serving as its precursor stage. Early intervention at the MCI stage can significantly delay AD onset.

This study employed untargeted urine metabolomics, with data obtained from the MetaboLights database (MTBLS8662), combined with orthogonal partial least squares-discriminant analysis (OPLS-DA) to examine metabolic differences across different stages of AD progression. A decision tree approach was used to identify key metabolites within significantly enriched pathways. These key metabolites were then utilized to construct and validate an AD progression prediction model.

The OPLS-DA model effectively distinguished the metabolic characteristics at different stages. Pathway enrichment analysis revealed that Drug metabolism was significantly enriched across all stages, while Retinol metabolism was particularly prominent during the transition stages. Key metabolites such as Theophylline, Vanillylmandelic Acid (VMA), and Adenosine showed significant differencesdifferencesin the early stages of the disease, whereas 1,7-Dimethyluric Acid, Cystathionine, and Indole exhibited strong predictive value during the MCI to AD transition. These metabolites play a crucial role in monitoring AD progression. Predictive models based on these metabolites demonstrated excellent classification and prediction capabilities.

This study systematically analyzed the dynamic metabolic differences during the progression of AD and identified key metabolites and pathways as potential biomarkers for early prediction and intervention. Utilizing urinary metabolomics, the findings provide a theoretical basis for monitoring AD progression and contribute to improving prevention and intervention strategies, thereby potentially delaying disease progression.

G-protein-coupled receptors (GPCRs) represent one of the largest classes of therapeutic targets. However, developing successful therapeutics to target GPCRs is a challenging endeavor, with many molecules failing during in vivo clinical trials due to a lack of efficacy. The in vitro identification of drug-target residence time (1/koff) has been suggested to improve predictions of in vivo success. Here, a ligand binding assay using fluorescence anisotropy was implemented to successfully determine on rates (kon) and off rates (koff) of labeled and unlabeled ligands binding to the adenosine A2A receptor (A2AR) purified into nanodiscs (A2AR-NDs). The kinetic assay was used to determine the optimal storage conditions of A2AR-NDs, where they were found to be stable for more than 6 months at -80°C. The binding assay was implemented to further understand receptor function by determining the effects of charged lipids on agonist binding kinetics, how sodium levels allosterically modulate A2AR function, and how A2AR protonation affects agonist binding.

Following transection, nerve repair using the polylactic acid (PLA) conduit is an effective option. In addition, inosine treatment has shown potential to promote nerve regeneration. Therefore, this study aimed to investigate the regenerative potential of inosine after nerve transection and polylactic acid conduit repair.

C57/Black6 mice were subjected to sciatic nerve transection, repair with PLA conduit, and intraperitoneal injection of saline or inosine 1 h after injury and daily for 1 week. To assess motor and sensory recovery, functional tests were performed before and weekly up to 8 weeks after injury. Following, to evaluate the promotion of regeneration and myelination, electroneuromyography, morphometric analysis and immunohistochemistry were then performed.

Our results showed that the inosine group had a greater number of myelinated nerve fibers (1,293 ± 85.49 vs. 817 ± 89.2), an increase in neurofilament high chain (NFH) and myelin basic protein (MBP) immunolabeling and a greater number of fibers within the ideal g-ratio (453.8 ± 45.24 vs. 336.6 ± 37.01). In addition, the inosine group presented a greater adenosine A2 receptor (A2AR) immunolabeling area. This resulted in greater compound muscle action potential amplitude and nerve conduction velocity, leading to preservation of muscle and neuromuscular junction integrity, and consequently, the recovery of motor and sensory function.

Our findings suggest that inosine may enhance regeneration and improve both motor and sensory function recovery after nerve transection when repaired with a poly-lactic acid conduit. This advances the understanding of biomaterials and molecular treatments.

The potential role of adenosine, a natural neuroprotective agent, and its receptors in the pathogenesis of Alzheimer's disease has been proposed. The present study aims to examine the effect of administering both an A1 receptor agonist and an A2A adenosine receptor antagonist simultaneously on memory, inflammatory factors, and PSD-95 in an LPS-induced Alzheimer's disease model in rats. Fifty-six male Wistar rats were randomly divided into seven groups: Saline, LPS, Saline + Vehicle, LPS + Vehicle, LPS + SCH58261 (A2A receptor antagonist), LPS + CPA (A1 receptor agonist), LPS + SCH58261+CPA. LPS (3 mg/kg/ip) was used to cause memory impairment. Treatment was performed by intraventricular injection of CPA at a dose of 700 μg and SCH-58261 at 40 μg for ten days. Passive avoidance and Y-maze tests were performed to examine animals' memories. IL-10, TNF-α, and PSD-95 levels were measured in the brain using ELISA and western blot, respectively. Compared to the groups receiving each medication separately, the simultaneous administration of CPA and SCH58261 improved memory (P<0.05). Additionally, compared to the single medication groups, there was a significant increase in IL-10, PSD-95, and a significant decrease in TNF-α in the brain tissue (P<0.05). These findings suggest that the activation of A1 receptors along with A2A receptor inhibition could be a potential therapeutic strategy for Alzheimer's disease. These findings suggest that A1 receptor activation combined with A2A receptor inhibition may be a promising therapeutic approach for Alzheimer's disease.

In males, adenosine (ADO) is known to relax penile smooth muscles, although its role in the vagina is not yet fully elucidated.

This study investigated the effect of ADO on vagina smooth muscle activity, using a validated female Sprague-Dawley rat model.

Contractility studies, using noradrenaline-precontracted vaginal strips, tested the effects of ADORA1/3 antagonists and ADORA2A/2B antagonists and agonists. Increasing doses of ADO were tested after in vivo or in vitro treatment with Nω-nitro-L-arginine-methyl-ester hydrochloride (L-NAME) or with guanylate or adenylate cyclase inhibitors. Immunopositivity for ADORA2A and ADORA2B was assessed, and messenger RNA (mRNA) analysis was performed. Cyclic ADO monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were quantified both in rat vagina smooth muscle cells (rvSMCs) and in vaginal tissues with increasing doses of ADO.

Demonstrating ADO's role in the relaxing/contractile mechanism in distal vagina smooth muscle.

All ADO receptors mRNAs were expressed in vaginal tissue, with a prevalent content of ADORA2B. A high expression of genes regulating ADO catabolism (ADK) and de novo synthesis (NT5E) was found. In vaginal strips, ADO induced relaxation with IC50 = 144.7 μM and a flat pseudo-Hill coefficient value = -0.42, indicating an activity on heterogeneous receptors. Blocking ADORA1/3 shifted ADO response to the left and with a steeper slope. ADORA2A/2B agonists showed a higher potency than ADO in inducing relaxation. Immunolocalization confirmed the presence of ADORA2A/2B in vaginal musculature, in the blood vessels endothelium, and in the epithelium. ADO stimulation of vagina tissues induced a significant increase in cAMP and cGMP contents. Experiments on rvSMCs confirmed that ADO time- and dose-dependently stimulated cAMP production in these cells. However, ADORA2A/2B antagonists, although reducing the ADO-induced relaxation, did not completely block it. A similar inhibition was obtained by blocking adenylate cyclase. Overall, these findings suggest that ADO relaxation involves other pathways, eg, nitric oxide (NO)/cGMP. Accordingly, blocking NO formation through L-NAME substantially blunted ADO responsiveness, as it does the block of cGMP formation through 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Simultaneous incubation with cGMP and cAMP blockers completely inhibited ADO responsiveness.

The study highlights ADO's role in regulating vaginal smooth muscle activity, suggesting its potential effect on the vagina.

This is the first study on ADO in the vagina, although the results are preliminary and limited to the rat model.

These results show that ADO acts as a vaginal relaxing modulator through selective activation of receptors involving not only cAMP but also cGMP.

Hyperbaric oxygen (HBO) refers to pure oxygen with a pressure greater than 1 atmospheres absolute (ATA), and when the pressure is too high, it can cause convulsive attacks. Adenosine and dopamine have been shown to be closely associated with HBO-induced convulsion seizures, and their receptors exhibited a coexisting relationship of mutual antagonism on the membrane of nerve cells. We explored the influence of adenosine and dopamine interplay on the occurrence of oxygen convulsion. Rats were individually exposed to HBO of 6 ATA and treated with adenosine, dopamine, and their receptor modulators separately and jointly, with the latency of convulsion onset recorded. In addition, after administering adenosine to rats and exposing them to HBO for 30 min, the content of dopamine and its metabolites and the activity of enzymes related to their metabolism were measured. The results revealed that dopamine was effective in resisting convulsion (>60 min vs. 32.53 ± 5.31 min, P = 0.000), and low-dose adenosine partially counteracted its effect (>60 min vs. 28.18 ± 6.24 min, P = 0.002). The combined use of adenosine A1 and dopamine D1 receptor modulators significantly impacted the incidence of convulsion. The activation or inhibition of the A2A receptor had a particularly significant impact on convulsion, whereas modulating the D2 receptor did not affect their effects. The combination of A1 agonist and D2 agonist was highly effective in resisting convulsion (>60 min vs. 32.53 ± 5.31 min, P = 0.000). Exposure to HBO accelerated the metabolism of dopamine to its end products, which may be related to the enhanced activity of monoamine oxidase (MAO). Adenosine can inhibit MAO activity (0.0766 ± 0.0150 U/mg.prot vs. 0.1055 ± 0.0086 U/mg.prot, P = 0.004), maintaining a higher level of dopamine (1.820 ± 0.379 mg/g vs. 0.602 ± 0.087 mg/g, P = 0.000). The study demonstrated that dopamine plays a significant role in oxygen convulsion and adenosine can affect dopamine metabolism. The interaction between them can have a crucial impact on the occurrence of oxygen convulsion. The findings offer a novel perspective for further investigating the mechanism of oxygen convulsion and exploring effective preventive strategies.NEW & NOTEWORTHY The interaction between adenosine and dopamine is critically important in determining the incidence of oxygen convulsion. Simultaneous regulation of both adenosine and dopamine offers a superior approach to counteract oxygen convulsion, achieving a synergistic effect exceeding the sum of their individual impacts. These findings provide new directions and insights for future in-depth and systematic exploration of the pathogenesis of central nervous system oxygen toxicity.

Deer mice (Peromyscus maniculatus bairdii), a wildtype species native to North America, have been investigated for their spontaneous compulsive-like behaviour. The repetitive and persistence nature of three unique compulsive-like phenotypes in deer mice, i.e., high stereotypy (HS), large nesting behaviour (LNB) and high marble burying (HMB), are characterized by behavioural and cognitive rigidity. In this narrative review, we summarize key advances in the model's application to study obsessive-compulsive disorder (OCD), emphasizing how it may be used to investigate neurobiological and neurocognitive aspects of rigidity. Indeed, deer mice provide the field with a unique naturalistic and spontaneous model system of behavioural and cognitive rigidity that is useful for investigating the psychobiological mechanisms that underpin a range of compulsive-like phenotypes. Throughout the review, we highlight new opportunities for future research.

Astrocytes participate in brain clearance of extracellular proteins and metabolites, through the activity of the water channel aquaporin-4 (AQP4), which can be deregulated in stress-related disorders, impairing brain waste clearance. The present study investigates the impact of dexamethasone (Dexa), a synthetic glucocorticoid used as a simplified in vitro stress model, on astrocytic AQP4 and its modulation by adenosine A2A receptors (A2AR), which blockade reverses conditions related with maladaptive stress, such as anxiety and depression. The clearance of proteins in primary astrocytic cultures, assessed using 5 kDa FITC-dextran and 45 kDa TRITC-dextran uptake, was decreased by a 24 h exposure to 100 nM Dexa. The Dexa exposure decreased α-syntrophin density, a protein-targeting AQP4 to astrocytic processes, potentially affecting AQP4 location and, consequently, its activity. Accordingly, Dexa exposure decreased astrocytic water influx (assessed with calcein fluorescence), which paralleled the impairment of dextran clearance. The Dexa-induced decrease in extracellular protein uptake was prevented by the AQP4 activator TGN-073 and A2AR antagonism with SCH58261, showing that the impairment of AQP4-mediated protein clearance was controlled by A2AR in this Dexa-simplified in vitro stress model. Additionally, the effects of Dexa in AQP4 location and activity were prevented by SCH58261, confirming that A2AR modulate AQP4 function. This conclusion was reinforced by the observed AQP4/A2AR physical interaction in astrocytes. Overall, the data indicate that in vitro conditions related to stress affect the localisation of astrocytic AQP4 and its role in extracellular protein uptake, which was modulated by A2AR. These findings unveil a novel therapeutic mechanism to prevent brain extracellular protein accumulation and associated neurological disorders by tinkering with AQP4 and A2AR.