A2AR-mediated CXCL5 upregulation on macrophages promotes NSCLC progression via NETosis.

Tumor-associated macrophages (TAMs) are abundant in tumors and interact with tumor cells, leading to the formation of an immunosuppressive microenvironment and tumor progression. Although many studies have explored the mechanisms underlying TAM polarization and its immunosuppressive functions, understanding of its progression remains limited. TAMs promote tumor progression by secreting cytokines, which subsequently recruit immunosuppressive cells to suppress the antitumor immunity. In this study, we established an in vitro model of macrophage and non-small cell lung cancer (NSCLC) cell co-culture to explore the mechanisms of cell-cell crosstalk. We observed that in NSCLC, the C-X-C motif chemokine ligand 5 (CXCL5) was upregulated in macrophages because of the stimulation of A2AR by adenosine. Adenosine was catalyzed by CD39 and CD73 in macrophages and tumor cells, respectively. Nuclear factor kappa B (NFκB) mediated the A2AR stimulation of CXCL5 upregulation in macrophages. Additionally, CXCL5 stimulated NETosis in neutrophils. Neutrophil extracellular traps (NETs)-treated CD8+ T cells exhibited upregulation of exhaustion-related and cytosolic DNA sensing pathways and downregulation of effector-related genes. However, A2AR inhibition significantly downregulated CXCL5 expression and reduced neutrophil infiltration, consequently alleviating CD8+ T cell dysfunction. Our findings suggest a complex interaction between tumor and immune cells and its potential as therapeutic target.

New comparative genomic evidence supporting the proteomic diversification role of A-to-I RNA editing in insects.

Adenosine-to-inosine (A-to-I) RNA editing, resembling A-to-G mutation, confers adaptiveness by increasing proteomic diversity in a temporal-spatial manner. This evolutionary theory named "proteomic diversifying hypothesis" has only partially been tested in very few organisms like Drosophila melanogaster, mainly by observing the positive selection on nonsynonymous editing events. To find additional genome-wide evidences supporting this interesting assumption, we retrieved the genomes of four Drosophila species and collected 20 deep-sequenced transcriptomes of different developmental stages and neuron populations of D. melanogaster. We systematically profiled the RNA editomes in these samples and performed meticulous comparative genomic analyses. Further evidences were found to support the diversifying hypothesis. (1) None of the nonsynonymous editing sites in D. melanogaster had ancestral G-alleles, while the silent editing sites had an unignorable fraction of ancestral G-alleles; (2) Only very few nonsynonymous editing sites in D. melanogaster had corresponding G-alleles derived in the genomes of sibling species, and the fraction of such situation was significantly lower than that of silent editing sites; (3) The few nonsynonymous editing with corresponding G-alleles had significantly more variable editing levels (across samples) than other nonsynonymous editing sites in D. melanogaster. The proteomic diversifying nature of RNA editing in Drosophila excludes the restorative role which favors an ancestral G-allele. The few fixed G-alleles in sibling species might facilitate the adaptation to particular environment and the corresponding nonsynonymous editing in D. melanogaster would introduce stronger advantage of flexible proteomic diversification. With multi-Omics data, our study consolidates the nature of evolutionary significance of A-to-I RNA editing sites in model insects.

A randomized controlled clinical trial examining the effects of Cordyceps militaris beverage on the immune response in healthy adults.

Cordyceps militaris (L.) Link (C. militaris) contains various beneficial substances, including polysaccharides (galactomannan), nucleotides (adenosine and cordycepin), cordycepic acid, amino acids, and sterols (ergosterol and beta-sitosterol). It also contains other essential nutrients, such as protein, vitamins (E, K, B1, B2, and B12), and minerals (potassium, sodium, calcium, magnesium, iron, zinc, and selenium). Due to the numerous health benefits of supplements and products containing C. militaris extract, their popularity has increased. However, the immunostimulant effect of C. militaris remains unclear. Therefore, this study developed a functional beverage from the submerged fermentation of C. militaris (FCM) and aimed to investigate the potential of FCM in healthy male and female volunteers in Phayao Province, Thailand. This study provides essential information for the development of healthy drink products. Healthy men and women were provided either FCM containing 2.85 mg of cordycepin or placebo for 8 weeks (n = 10 for each gender). The immune cell markers, immunoglobulins, and safety parameters were assessed initially at baseline and at 4 and 8 weeks. The NK cell activity markedly increased in the male FCM group from baseline (p = 0.049) to 4 weeks after receiving FCM. Compared with those in the placebo group, the NK activity in women who received FCM for 8 weeks significantly increased (p = 0.023) from baseline. Within-group analysis revealed that the IL-1ß levels were markedly reduced in the male FCM group (p = 0.049). Furthermore, the IL-6 levels decreased from baseline in the female FCM group (p = 0.047). The blood sugar, lipid, and safety indices were not different between the experimental groups. FCM can potentially be developed as an immune-boosting supplement without liver, kidney, or blood component toxicity.

Structural and functional effects of inosine modification in mRNA.

Inosine (I), resulting from the deamination of adenosine (A), is a prominent modification in the human transcriptome. The enzymes responsible for the conversion of adenosine to inosine in human mRNAs are the ADARs (adenosine deaminases acting on RNA). Inosine modification introduces a layer of complexity to mRNA processing and function, as it can impact various aspects of RNA biology, including mRNA stability, splicing, translation, and protein binding. The relevance of this process is emphasized in the growing number of human disorders associated with dysregulated A-to-I editing pathways. Here, we describe the impact of the A-to-I conversion on the structure and stability of duplex RNA and on the consequences of this modification at different locations in mRNAs. Furthermore, we highlight specific open questions regarding the interplay between inosine formation in duplex RNA and the innate immune response.

Purinergic enzymes on extracellular vesicles: immune modulation on the go.

An increase in the extracellular concentration of ATP as a consequence of cellular stress or cell death results in the activation of immune cells. To prevent inflammation, extracellular ATP is rapidly metabolized to adenosine, which deploys an anti-inflammatory signaling cascade upon binding to P1 receptors on immune cells. The ectonucleotidases necessary for the degradation of ATP and generation of adenosine are present on the cell membrane of many immune cells, and their expression is tightly regulated under conditions of inflammation. The discovery that extracellular vesicles (EVs) carry purinergic enzyme activity has brought forward the concept of EVs as a new player in immune regulation. Adenosine-generating EVs derived from cancer cells suppress the anti-tumor response, while EVs derived from immune or mesenchymal stem cells contribute to the restoration of homeostasis after infection. Here we will review the existing knowledge on EVs containing purinergic enzymes and molecules, and discuss the relevance of these EVs in immune modulation and their potential for therapy.

Transcriptome analysis of cultured human vascular endothelial cells after γ-ray irradiation and correlation analysis with ATP, ADP, and adenosine as secondary soluble factors.

Vascular endothelial cells serve as barriers between blood components and subendothelial tissue and regulate the blood coagulation-fibrinolytic system. Ionizing radiation is a common physical stimulant that induces a bystander effect whereby irradiated cells influence neighboring cells through signalings, including purinergic receptor signaling, activated by adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine as secondary soluble factors. Human vascular endothelial EA.hy926 cells were cultured and irradiated with γ-rays or treated with ATP, ADP, or adenosine under non-toxic conditions. RNA-seq, gene ontology, and hierarchical clustering analyses were performed. The transcriptome analysis of differentially expressed genes in vascular endothelial cells after γ-ray irradiations suggests that the change of gene expression by γ-irradiation is mediated by ATP and ADP. In addition, the expression and activity of the proteins related to blood coagulation and fibrinolysis systems appear to be secondarily regulated by ATP and ADP in vascular endothelial cells after exposure to γ-irradiation. Although it is unclear whether the changes of the gene expression related to blood coagulation and fibrinolysis systems by γ-irradiation affected the increased hemorrhagic tendency through the exposure to γ-irradiation or the negative feedback to the activated blood coagulation system, the present data indicate that toxicity associated with γ-irradiation involves the dysfunction of vascular endothelial cells related to the blood coagulation-fibrinolytic system, which is mediated by the signalings, including purinergic receptor signaling, activated by ATP and ADP.

Structure, function and substrate preferences of archaeal S-adenosyl-L-homocysteine hydrolases.

S-Adenosyl-L-homocysteine hydrolase (SAHH) reversibly cleaves S-adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine-dependent methylation reactions. The conversion of S-adenosyl-L-homocysteine into adenosine and L-homocysteine plays an important role in the regulation of the methyl cycle. An alternative metabolic route for S-adenosyl-L-methionine regeneration in the extremophiles Methanocaldococcus jannaschii and Thermotoga maritima has been identified, featuring the deamination of S-adenosyl-L-homocysteine to S-inosyl-L-homocysteine. Herein, we report the structural characterisation of different archaeal SAHHs together with a biochemical analysis of various SAHHs from all three domains of life. Homologues deriving from the Euryarchaeota phylum show a higher conversion rate with S-inosyl-L-homocysteine compared to S-adenosyl-L-homocysteine. Crystal structures of SAHH originating from Pyrococcus furiosus in complex with SLH and inosine as ligands, show architectural flexibility in the active site and offer deeper insights into the binding mode of hypoxanthine-containing substrates. Altogether, the findings of our study support the understanding of an alternative metabolic route for S-adenosyl-L-methionine and offer insights into the evolutionary progression and diversification of SAHHs involved in methyl and purine salvage pathways.

Phosphoglycerate Kinase 1: An Effective Therapeutic Target in Cancer.

Phosphoglycerate kinase 1 (PGK1) serves as a pivotal enzyme in the cellular glycolysis pathway, facilitating adenosine-triphosphate (ATP) production in tumor cells and driving the Warburg effect. PGK1 generates ATP through the reversible phosphorylation reaction of 1,3-bisphosphoglycerate (1,3-BPG) to Mg-adenosine-5'-diphosphate (Mg-ADP). In addition to its role in regulating cellular metabolism, PGK1 plays a pivotal role in autophagy induction, regulation of the tricarboxylic acid cycle (TCA), and various mechanisms including tumor cell drug resistance, and so on. Given its multifaceted functions within cells, the involvement of PGK1 in many types of cancer, including breast cancer, astrocytoma, metastatic colon cancer, and pancreatic ductal adenocarcinoma, is intricate. Notably, PGK1 can function as an intracellular protein kinase to coordinate tumor growth, migration, and invasion via posttranslational modifications (PTMs). Furthermore, elevated expression levels of PGK1 have been observed in cancer tissues, indicating its association with unfavorable treatment outcomes and prognosis. This review provides a comprehensive summary of PGK1's expression pattern, structural features, functional properties, involvement in PTMs, and interaction with tumors. Additionally highlighted are the prospects for developing and applying related inhibitors that confirm the indispensable value of PGK1 in tumor progression.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

18F-Labeled PET Tracers Specific for Adenosine A2A Receptor: Design, Synthesis, and Biological Evaluation.

By modifying the structures of targeted A2AR antagonists and tracers, novel compounds 3, 7a, 9, 12c, and BIBD-399 were designed and synthesized. In vitro inhibition experiments demonstrated that 3, 12c, and BIBD-399 have high affinity for A2AR. [18F]3 and [18F]BIBD-399 were successfully synthesized. In terms of biological distribution, the brain uptake of [18F]MNI-444 exhibits greater than that of [18F]3 and [18F]BIBD-399. PET imaging shows that [18F]3 is off-target in the brain, while [18F]BIBD-399 and [18F]MNI-444 can be specifically imaged in regions with high A2AR expression. Differently, [18F]BIBD-399 could quickly reach equilibrium in the targeted region within 10 min after administration, while [18F]MNI-444 shows a slowly increasing trend within 2 h of administration. [18F]BIBD-399 is mainly metabolized by the liver and kidney, and there is no obvious defluorination in vivo. Additional in vitro autoradiography showed that the striatal signals of [18F]BIBD-399 and [18F]MNI-444 were inhibited by the A2AR antagonist SCH442416 but not by the A1R antagonist DPCPX, demonstrating the high A2AR binding specificity of [18F]BIBD-399. Molecular docking further confirms the high affinity of MNI-444 and BIBD-399 for A2AR. Further tMCAo imaging showed that [18F]BIBD-399 can sensitively distinguish between infarcted and noninfarcted sides, a capability not observed with [18F]MNI-444. Given its pharmacokinetic properties and the ability to identify lesion regions, [18F]BIBD-399 has potential advantages in monitoring A2AR changes, meriting further clinical investigation.

An adenosine derivative promotes mitochondrial supercomplexes reorganization and restoration of mitochondria structure and bioenergetics in a diethylnitrosamine-induced hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) progression is associated with dysfunctional mitochondria and bioenergetics impairment. However, no data about the relationship between mitochondrial supercomplexes (hmwSC) formation and ATP production rates in HCC are available. Our group has developed an adenosine derivative, IFC-305, which improves mitochondrial function, and it has been proposed as a therapeutic candidate for HCC. We aimed to determine the role of IFC-305 on both mitochondrial structure and bioenergetics in a sequential cirrhosis-HCC model in rats. Our results showed that IFC-305 administration decreased the number and size of liver tumors, reduced the expression of tumoral markers, and reestablished the typical architecture of the hepatic parenchyma. The livers of treated rats showed a reduction of mitochondria number, recovery of the mtDNA/nDNA ratio, and mitochondrial length. Also, IFC-305 increased cardiolipin and phosphatidylcholine levels and promoted hmwSC reorganization with changes in the expression levels of hmwSC assembly-related genes. IFC-305 in HCC modified the expression of several genes encoding elements of electron transport chain complexes and increased the ATP levels by recovering the complex I, III, and V activity. We propose that IFC-305 restores the mitochondrial bioenergetics in HCC by normalizing the quantity, morphology, and function of mitochondria, possibly as part of its hepatic restorative effect.

Adenosine A2B receptor agonist improves epidermal barrier integrity in a murine model of epidermal hyperplasia.

Adenosine regulates multiple physiological processes through the activation of four receptor subtypes, of which the A2B adenosine receptor (A2BAR) has the lowest affinity for adenosine. Being the adenosine receptor subtype most prominently expressed in epidermis, we recently described the antiproliferative and anti-inflammatory effect of the selective A2BAR agonist BAY60-6583 (BAY) in human keratinocytes stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), so we sought to establish the effect of topical application of BAY in a model of murine epidermal hyperplasia. Topical application of BAY (1 or 10 µg/site) prevented the inflammatory reaction and skin lesions induced by TPA, minimizing hyperproliferation and acanthosis, as well as the expression of specific markers of proliferative keratinocytes. On the other hand, pre-treatment with the selective A2BAR antagonist, PSB-1115 (PSB, 5 or 50 µg/site) reversed these beneficial effects. Additionally, BAY application normalized the expression of epidermal barrier proteins, whose integrity is altered in inflammatory skin diseases, while treatment with the antagonist alone worsened it. Our results, besides confirming the anti-inflammatory and antiproliferative effects of the A2BAR agonist, further demonstrate a role of A2BAR activation to preserve the epidermal barrier. Therefore, the activation of A2BAR may constitute a possible new pharmacological target for the treatment of skin inflammatory diseases such as psoriasis.

Sexual stage-specific A-to-I mRNA editing is mediated by tRNA-editing enzymes in fungi.

A-to-I RNA editing catalyzed by adenosine-deaminase-acting-on-RNA (ADARs) was assumed to be unique to metazoans because fungi and plants lack ADAR homologs. However, genome-wide messenger RNA (mRNA) editing was found to occur specifically during sexual reproduction in filamentous ascomycetes. Because systematic characterization of adenosine/cytosine deaminase genes has implicated the involvement of TAD2 and TAD3 orthologs in A-to-I editing, in this study, we used genetic and biochemical approaches to characterize the role of FgTAD2, an essential adenosine-deaminase-acting-on-tRNA (ADAT) gene, in mRNA editing in Fusarium graminearum. FgTAD2 had a sexual-stage-specific isoform and formed heterodimers with enzymatically inactive FgTAD3. Using a repeat-induced point (RIP) mutation approach, we identified 17 mutations in FgTAD2 that affected mRNA editing during sexual reproduction but had no effect on transfer RNA (tRNA) editing and vegetative growth. The functional importance of the H352Y and Q375*(nonsense) mutations in sexual reproduction and mRNA editing were confirmed by introducing specific point mutations into the endogenous FgTAD2 allele in the wild type. An in vitro assay was developed to show that FgTad2-His proteins purified from perithecia, but not from vegetative hyphae, had mRNA editing activities. Moreover, the H352Y mutation affected the enzymatic activity of FgTad2 to edit mRNA but had no effect on its ADAT activity. We also identified proteins co-purified with FgTad2-His by mass spectrometry analysis and found that two of them have the RNA recognition motif. Taken together, genetic and biochemical data from this study demonstrated that FgTad2, an ADAT, catalyzes A-to-I mRNA editing with the stage-specific isoform and cofactors during sexual reproduction in fungi.

Subtle Structural Changes across the Boundary between A2AR/A2BR Dual Antagonism and A2BR Antagonism: A Novel Class of 2-Aminopyrimidine-Based Derivatives.

Aberrantly elevated adenosine in the tumor microenvironment exerts its immunosuppressive functions through adenosine receptors A2AR and A2BR. Antagonism of A2AR and A2BR has the potential to suppress tumor growth. Herein, we report a systemic assessment of the effects of an indole modification at position 4, 5, 6, or 7 on both A2AR/A2BR activity and selectivity of novel 2-aminopyrimidine compounds. Substituting indole at the 4-/5-position produced potent A2AR/A2BR dual antagonism, whereas the 6-position of indole substitution gave highly selective A2BR antagonism. Molecular dynamics simulation showed that the 5-cyano compound 7ai had a lower binding free energy than the 6-cyano compound 7aj due to water-bridged hydrogen bond interactions with E169 or F168 in A2AR. Of note, dual A2AR/A2BR antagonism by compound 7ai can profoundly promote the activation and cytotoxic function of T cells. This work provided a strategy for obtaining novel dual A2AR/A2BR or A2BR antagonists by fine-tuning structural modification.

Epitope Mapping of an Anti-Mouse CD39 Monoclonal Antibody Using PA Scanning and RIEDL Scanning.

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular adenosine triphosphate into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (anti-mCD39) mAb, C39Mab-1 using the Cell-Based Immunization and Screening method. In this study, we determined the critical epitope of C39Mab-1 using flow cytometry. We performed the PA tag (12 amino acids [aa])-substituted analysis (named PA scanning) and RIEDL tag (5 aa)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275-279, 282-291, and 306-323 aa of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

Dysregulation in CD39/CD73 Axis May Trigger the Upsurge of the Immune Suppressive Agent Adenosine in OSA Patients.

INTRODUCTION: Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Interestingly, adenosinergic signaling has emerged as a powerful immune checkpoint driving tumor development and progression. METHODS: Here, we explored the expression of the adenosinergic ecto-enzymes CD39 and CD73 in T-lymphocytes of OSA patients without any evidence of cancer, as well as their soluble forms in plasma (sCD39 and sCD73), along with adenosine. In addition, we explored the role of intermittent hypoxia (IH) in this context by in vitro models. RESULTS: Our results showed that CD39 is upregulated while CD73 is downregulated in OSA T-cells' membrane. Moreover, our findings suggest that IH, through HIF-1, mediates the upregulation of both CD39 and CD73; and that CD73 downregulation could be mediated by a higher release of sCD73 by OSA T-lymphocytes. Importantly, we found that both sCD39 and sCD73 are upregulated in OSA plasma, suggesting T-lymphocytes as a potential source for plasmatic sCD73. Finally, our data propose the alterations in CD39/CD73 axis could underlie the upsurge of adenosine levels in the plasma of OSA patients. CONCLUSION: Our study reveals a hypoxia-mediated alteration of the CD39/CD73 axis in OSA patients, which could trigger ADO upregulation, thus potentially contributing to the immune suppressive environment and ultimately facilitating tumor development and progression. Therefore, our data highlights the need for new longitudinal studies evaluating CD39 and/or CD73 as potential cancer-risk prognostic biomarkers in OSA patients.

Feedback activation of CD73-Adenosine axis attenuates the antitumor immunity of STING pathway.

The cGAS-STING pathway, a crucial component of innate immunity, has garnered attention as a potential therapeutic target for tumor treatment, but targeting this pathway is complicated by diverse feedback mechanisms of the cGAS-STING pathway. In this study, we demonstrated that STING activation enhanced the expression of CD73 and the subsequent production of adenosine in immune cells and cancer cells. Mechanistically, the feedback activation of CD73 depended on the type I IFN/IFNAR axis induced by STING activation. Furthermore, the combination of STING agonist and anti-CD73 mAb markedly blocked tumor growth in vivo by promoting the infiltration of CD8+ T cells and reducing the accumulation of Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment. Our work provides a rationale for the combination of STING agonists and CD73 inhibitors in cancer immunotherapy.

Epitranscriptomic regulation in fasting hearts: implications for cardiac health.

Cardiac tolerance to ischaemia can be increased by dietary interventions such as fasting, which is associated with significant changes in myocardial gene expression. Among the possible mechanisms of how gene expression may be altered are epigenetic modifications of RNA - epitranscriptomics. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two of the most prevalent modifications in mRNA. These methylations are reversible and regulated by proteins called writers, erasers, readers, and m6A-repelled proteins. We analysed 33 of these epitranscriptomic regulators in rat hearts after cardioprotective 3-day fasting using RT-qPCR, Western blot, and targeted proteomic analysis. We found that the most of these regulators were changed on mRNA or protein levels in fasting hearts, including up-regulation of both demethylases - FTO and ALKBH5. In accordance, decreased methylation (m6A+m6Am) levels were detected in cardiac total RNA after fasting. We also identified altered methylation levels in Nox4 and Hdac1 transcripts, both of which play a role in the cytoprotective action of ketone bodies produced during fasting. Furthermore, we investigated the impact of inhibiting demethylases ALKBH5 and FTO in adult rat primary cardiomyocytes (AVCMs). Our findings indicate that inhibiting these demethylases reduced the hypoxic tolerance of AVCMs isolated from fasting rats. This study showed that the complex epitranscriptomic machinery around m6A and m6Am modifications is regulated in the fasting hearts and might play an important role in cardiac adaptation to fasting, a well-known cardioprotective intervention.

Tumor cell senescence-induced macrophage CD73 expression is a critical metabolic immune checkpoint in the aging tumor microenvironment.

Background: The role of senescent cells in the tumor microenvironment (TME) is usually bilateral, and diverse therapeutic approaches, such as radiotherapy and chemotherapy, can induce cellular senescence. Cellular interactions are widespread in the TME, and tumor cells reprogram immune cells metabolically by producing metabolites. However, how senescent cells remodel the metabolism of TME remains unclear. This study aimed to explore precise targets to enhance senescent cells-induced anti-tumor immunity from a metabolic perspective. Methods: The in vivo senescence model was induced by 8 Gy×3 radiotherapy or cisplatin chemotherapy, and the in vitro model was induced by 10 Gy-irradiation or cisplatin treatment. Metabonomic analysis and ELISA assay on tumor interstitial fluid were performed for metabolites screening. Marker expression and immune cell infiltration in the TME were analyzed by flow cytometry. Cell co-culture system and senescence-conditioned medium were used for crosstalk validation in vitro. RNA sequencing and rescue experiments were conducted for mechanism excavation. Immunofluorescence staining and single-cell transcriptome profiling analysis were performed for clinical validation. Results: We innovatively reveal the metabolic landscape of the senescent TME, characterized with the elevation of adenosine. It is attributed to the senescent tumor cell-induced CD73 upregulation of tumor-associated macrophages (TAMs). CD73 expression in TAMs is evoked by SASP-related pro-inflammatory cytokines, especially IL-6, and regulated by JAK/STAT3 pathway. Consistently, a positive correlation between tumor cells senescence and TAMs CD73 expression is identified in lung cancer clinical specimens and databases. Lastly, blocking CD73 in a senescent background suppresses tumors and activates CD8+ T cell-mediated antitumor immunity. Conclusions: TAMs expressed CD73 contributes significantly to the adenosine accumulation in the senescent TME, suggesting targeting CD73 is a novel synergistic anti-tumor strategy in the aging microenvironment.

Biochemical evaluation and ligand binding studies on glycerophosphodiester phosphodiesterase from Staphylococcus aureus using STD-NMR spectroscopy and molecular docking analysis.

Glycerophosphodiester phosphodiesterase (GDPD) is a highly conserved enzyme in both prokaryotic and eukaryotic organisms. It catalyses the hydrolysis of various glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols, which serve as building blocks in several biosynthetic pathways. This enzyme is a well-known virulence factor in many pathogenic bacteria, including Staphylococcus aureus, and is thus considered a potential drug target. In this study, competent E. coli BL21(DE3)pLysS expression cells were used to express the GDPD enzyme from vancomycin-resistant Staphylococcus aureus (VRSA), which was then purified using size exclusion and anion exchange chromatography. The hydrolytic activity of GDPD was evaluated on the non-physiological substrate bis(p-nitrophenyl) phosphate (BpNPP), which indicated functional activity of the enzyme. 79 drugs were evaluated for their inhibitory potential against GDPD enzyme by the colorimetric assay. Out of 79 drugs, 13 drugs, including tenofovir (1), adenosine (2), clioquinol (11), bromazepam (12), lamotrigine (13), sulfadiazine (14), azathioprine (15), nicotine (16), sitagliptin PO4 (17), doxofylline (18), clindamycin phosphate (19), gentamycin sulphate (20), and ceftriaxone sodium (21) revealed varying degrees of inhibitory potential with IC50 values in the range of 400 ± 0.007-951 ± 0.016 µM. All drugs were also evaluated for their binding interactions with the target enzyme by saturation transfer difference (STD-NMR) spectroscopy. 10 drugs demonstrated STD interactions and hence, showed binding affinity with the enzyme. Exceptionally, tenofovir (1) was identified to be a better inhibitor with an IC50 value of 400 ± 0.007 µM, as compared to the standard EDTA (ethylenediaminetetraacetic acid) (IC50 = 470 ± 0.008 µM). Moreover, molecular docking studies have identified key interactions of the ligand (tenofovir) with the binding site residues of the enzyme.