Adenosine regulates neurotransmission, immunity, inflammation, and tissue protection via four G protein-coupled receptors, among which the adenosine A3 receptor (A3R) remains comparatively understudied despite its therapeutic promise. This study examined whether cannabidiol (CBD) and cannabigerol (CBG) modulate human A3R signalling, alone or with added adenosine, using cAMP assays, molecular docking, binding-energy calculations, and molecular dynamics simulations. Both cannabinoids inhibited forskolin-stimulated cAMP accumulation in A3R-expressing cells, an effect absent in non-transfected cells and blocked by the selective A3R antagonist PSB-10 but not by the selective A2A receptor antagonist SCH 58261, supporting A3R-mediated activity under these conditions. CBG was more potent than CBD (IC50 = 0.130 vs. 1.05µM), whereas their maximal inhibitory responses were comparable. In the presence of 100nM CBD, the apparent IC50 of adenosine decreased from 138 to 42nM, whereas 100nM CBG reduced it to 1.0nM, corresponding to an approximately 138-fold increase in apparent adenosine potency; CBG also increased the maximal inhibitory response. These results identify CBG as an effective enhancer of adenosine potency at human A3R, suggesting it stabilizes an occupancy-dependent secondary pose that fine-tunes the orthosteric agonist-receptor interaction network. Further work, encompassing direct binding studies, proximal signalling assays, receptor-subtype profiling, and structural analyses, is needed to validate this mechanism.

Poly(A) tail plays critical roles in mRNA stability and translation efficiency, enhancing the potential of mRNA in vaccine and therapeutics. Encoded poly(dA) tail provides advantages over enzymatically synthesized tails, including product homogeneity, higher reproducibility, and reduced manufacturing costs. However, instability of homopolymeric regions during E. coli replication often leads to truncations, making accurate measurement of poly(dA) length essential for process optimization. Traditional methods such as Sanger sequencing are limited by labor-intensive workflows and low resolution of heterogeneous populations. Here, we developed a liquid chromatography-mass spectrometry (LC-MS) workflow to characterize encoded poly(dA) tail length distributions in plasmid DNA. In this study, two sample collection methods, polymerase chain reaction (PCR) and restriction enzyme digestion, were evaluated for LC-MS analysis. Both strategies achieved single-adenosine resolution and confirmed theoretical poly(dA) lengths. The PCR method required lower sample input but introduced shorter poly(dA) populations due to polymerase slippage, resulting in broader distributions. In contrast, the digestion method provided higher accuracy through an amplification-free process but required higher sample input and was limited by enzyme recognition sites. Agarose gel electrophoresis was applied as a preliminary assessment tool to estimate heterogeneity, while LC-MS enabled precise distribution profiling. The polydispersity index (PDI) was introduced to quantify tail heterogeneity, and the PDI values obtained from the two sample collection methods for LC-MS analysis showed the same trend as Sanger sequencing for stable and unstable poly (dA) tails. Overall, LC-MS combined with enzymatic digestion provides an accurate approach for investigating encoded poly(dA) length distribution, supporting mRNA drug development and quality control.

Cardiac fibrosis independently predicts adverse outcomes in heart failure (HF), yet no Food and Drug Administration-approved therapy directly targets fibrotic remodeling in the heart. To address this unmet clinical need, we used a multidimensional drug discovery pipeline centered on a human induced pluripotent stem cell (iPSC)-based platform. Through high-throughput screening, we identified CGS15943 (CGS) as the lead antifibrotic compound and validated its activity in human cardiac fibroblasts, three-dimensional engineered heart tissues, and animal models of HF. Mechanistic studies revealed an atypical adenosine receptor (AR)-dependent signaling pathway in which AR subtypes converge on Gβγ (the βγ subunits of heterotrimeric GTP-binding proteins) to activate phosphoinositide 3-kinase (PI3K)-AKT and yes-associated protein (YAP). CGS suppressed this signaling axis, thereby reducing fibrotic gene expression and fibroblast activation. These findings establish AR-driven Gβγ signaling as a potential therapeutic target for cardiac fibrosis.

N6-methyladenosine (m6A), one of the most abundant chemical modifications on RNA, is installed by METTL3 and several other methyltransferases, including METTL16. Although METTL16 has been characterized in several model organisms, its function in Drosophila remains unknown. Here, we show that, unlike in mammals, Drosophila Mettl16 mutants are viable but exhibit multiple developmental and behavioral defects. Both male and female mutants are sterile, with severe gametogenesis defects. Mettl16 mutant germ cells can pass the mitotic and meiotic stages, but are defective in spermatid elongation and individualization. Mettl16-GFP localizes predominantly to the nucleus, and its absence does not impair global protein synthesis in wing disc epithelial cells. MeRIP-Seq analysis indicates that Mettl16 loss affects m6A on only a small subset of transcripts, in contrast to the broad effect of Mettl3. We further demonstrate that Mettl16 interacts with U6 snRNA and is required for its m6A modification. Accordingly, Mettl16 mutants display widespread alterations in alternative splicing. Together, the pleiotropic phenotypes of Mettl16 mutants likely stem from its role in m6A deposition and U6-dependent splicing regulation of specific target transcripts.

Vespa basalis Smith venom (VBsV) causes painful and potentially severe systemic reactions, yet the relationships among its composition, bioactivity, and cytotoxicity remain poorly understood. Proteomic and metabolomic profiling was combined with ultrafiltration and gel filtration to characterize VBsV and obtain >3 kDa, operationally defined 700-3000 Da, and <700 Da fractions. The effect on LPS-induced NO production was evaluated by nitric oxide (NO) release in LPS-stimulated RAW264.7 cells, and cytotoxicity was assessed in tumor and THLE-2 liver cells. Five venom allergen proteins were identified, including two phospholipase A1 isoforms, hyaluronidase A, antigen 5, and venom dipeptidyl peptidase IV. Targeted profiling also detected several neurotransmitters and energy metabolites, including 5-hydroxytryptamine, L-glutamate, histamine, γ-aminobutyric acid, inosine, and adenosine. The 700-3000 Da fraction exhibited the strongest NO-suppressive and tumor-cell-viability effects among the tested fractions. The 700-3000 Da fraction suppressed NO release (IC50, approximately 23 μg/mL) and reduced tumor-cell viability, while minimally affecting THLE-2 cells at 10-80 μg/mL; by contrast, the >3 kDa fraction showed greater THLE-2 cytotoxicity. These findings identify the 700-3000 Da fraction as a priority for direct compositional characterization and isolation of the responsible molecules.

Cordyceps militaris is an edible entomopathogenic fungus with substantial nutritional and medicinal value. However, the molecular mechanisms underlying the vegetative mycelia-to-primordia (PR) transition and bioactive metabolite synthesis are still poorly defined. Deletion of the acyl-CoA dehydrogenase gene (ACAD) caused malformed, stunted PR-like structures together with excessive aerial mycelial growth, severely disrupting PR initiation and fruiting-body elongation in C. militaris. Transcriptomic, untargeted metabolomic and multi-omics analyses were performed on wild-type (WT) and ACAD-knockout strains at the mycelial and PR stages to explore gene expression reprogramming and bioactive nucleoside accumulation during the developmental transition and under ACAD deficiency. In total, 2014, 448, and 838 differentially expressed genes (DEGs) were identified in WT PR vs. WT mycelia, ΔACAD mycelia vs. WT mycelia, and ΔACAD PR vs. WT PR, demonstrating extensive transcriptional reprogramming during the development switch and upon ACAD deletion. KEGG enrichment and WGCNA analyses showed that ACAD deficiency significantly suppresses multiple reproductive-development pathways, including cell cycle progression, purine biosynthesis, the TCA cycle, MAPK signaling, redox homeostasis, fatty acid and amino acid metabolism. Consistently, ACAD deletion markedly increased intracellular ROS level and substantially reduced cellular ATP levels in mycelia. Several crucial genes modulated by ACAD were identified, including GTP cyclohydrolase I, histone lysine N-methyltransferase ASHR1, adenine phosphoribosyl transferase 1, glutathione-S-transferase F3 and CoA-ligase CCL8. Multi-omics integration demonstrated tightly coordinated transcriptional and metabolic regulation dependent on ACAD function. ACAD deficiency significantly disturbed amino acid, lipid, and nucleotide metabolism and reduced the abundance of amino acid derivatives, lipid compounds and purine nucleoside precursors in mycelia. Particularly, adenosine analogs serve as key precursors of the core bioactive nucleoside cordycepin. Key DEGs, including peroxisomal (S)-2-hydroxyacid oxidase, isocitrate lyase 2 and L-ascorbate peroxidase 5, were linked to these metabolic shifts. Our findings demonstrate that ACAD serves as an essential regulator that interconnects carbon metabolism, redox homeostasis, lipid, amino acid and purine metabolism to promote PR formation and secondary metabolite synthesis in C. militaris.

A-to-I RNA editing is increasingly recognized as a regulator of fungal development and pathogenesis, yet its extent and lifecycle dependence remain poorly characterized in basidiomycetes. Microbotryum superbum (MvSup), M. intermedium (MI), and M. lychnidis-dioicae (MVLG) are members of the M. violaceum fungal complex. Each species infects specific host plant species, resulting in commonly anther-smut disease. The lifecycle of these basidiomycete fungi includes the haploid, mating, and infection stages. RNA editing is a post-transcriptional process where adenosine (A) is converted to inosine (I) by adenosine deaminase enzymes, and such modifications to RNAs may lead to synonymous and nonsynonymous codon changes, thereby altering protein function. Here, we compared A-to-I editing across the haploid, mating, and infection stages of these three related species to determine how editing patterns vary with lifecycle stage and among closely related fungal pathogens. The a2 haploid strain of MI had fewer editing sites compared to other haploid strains. The predicted codon/amino acid changes in each haploid strain across the three species indicated three primary types of resulting amino acid substitutions that were common to both of the mating-type strains across the three species: threonine to alanine, lysine to glutamic acid, and valine to alanine. During the mating stage of MvSup, a synonymous codon change was found in a mitogen-activated protein kinase domain-containing protein within the protein's conserved region. Gene expression analysis revealed that certain genes, uniquely edited during the mating stage of MvSup, tend to be upregulated in the haploid stage but downregulated during mating, and vice versa. Research on RNA editing in basidiomycetes is relatively new. RNA editing mechanisms in fungi have been implicated in fungal pathogenesis, although the exact roles and implications remain unclear. Additional research will help us understand the functional significance of this apparently ubiquitous process in several members of the Microbotryum fungal complex, with possible ramifications more generally in fungi.

Mammalian Na+/Ca2+ exchangers (NCXs) mediate inward (Ca2+-exit) or outward (Ca2+-entry) currents (INCX), with the peak current (Ipeak) declining to the steady-state current (Iss). The Ca2+-, ATP-, and PIP2-dependent activation of cardiac (NCX1.1) inward INCX has been previously documented. To investigate possible interactions among regulatory modes, we examined the effects of ATP and PIP2 on outward INCX when the CBD1 and CBD2 domains of NCX1.4 (brain variant) are Ca2+-occupied and -unoccupied. At nominally zero cytosolic Ca2+, ATP and PIP2 significantly reduced the outward Ipeak amplitude. While increasing cytosolic Ca2+ increased Ipeak and Iss/Ipeak, consistent with CBD1- and CBD2-dependent regulation, no significant effects of ATP or PIP2 were detected at 0.1-10 μM cytosolic Ca2+. These observations indicate distinct responses under the Ca2+ conditions examined and warrant direct mechanistic investigation. Although we observed opposing effects of ATP and PIP2 on inward (in NCX1.1) and outward (in NCX1.4) currents using different patch-clamp configurations and protocols, the data collectively suggest that Ca2+, ATP, and PIP2 regulatory modes may operate differently on the forward and reverse modes in NCX1.1 and NCX1.4 variants. Given the important physiological implications across distinct cell types (e.g., cardiomyocytes, neurons, glia, and kidney), careful investigation is needed to resolve how distinct regulatory modes affect the forward and reverse modes of mammalian NCX variants.

Age-related decline in learning and memory functions poses significant challenges in an aging society, with epigenetic dysregulation emerging as a key contributor to cognitive deterioration. As the most prevalent internal RNA modification, N6-methyladenosine (m6A) dynamically orchestrates neural transcriptome plasticity through its "writers," "erasers," and "readers," yet its role in aging-associated cognitive impairment remains underexplored. This study employs an integrated epitranscriptomic approach to investigate m6A-mediated regulation in hippocampal aging processes. Through comparative m6A-mRNA epitranscriptomic microarray analysis of senescence-accelerated mouse prone 8 (SAMP8) and senescence-resistant SAMR1 hippocampi, we identified neural cell adhesion molecule 1 (NCAM1) as a key m6A-regulated effector whose decreased expression correlates with accelerated cognitive deterioration. Mechanistically, we revealed that Methyltransferase-like 3 (METTL3)-mediated m6A modification governs Ncam1 mRNA stability through insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) reader protein-dependent mechanisms, forming a regulatory axis that modulates cyclic AMP response element-binding protein (CREB) signaling pathway activity. Remarkably, targeting of this METTL3/IGF2BP1/NCAM1 axis significantly attenuated cognitive deficits in aged SAMP8 mice. Our findings establish an m6A methylation-dependent paradigm for NCAM1-mediated cognitive preservation during aging, uncovering a novel epitranscriptomic layer in age-related neurodegeneration.

Heavy-metal ATPase (HMA) is widely involved in the absorption and transport of metal elements, including copper, zinc, and cadmium in plants. Although the characterization of HMA has been conducted in several plants, the research on the HMA gene in Stevia rebaudiana remains limited. In this study, twelve SrHMA genes were identified, encoding stable proteins mostly localized to the plasma membrane and possessing conserved E1-E2_ATPase and hydrolase domains, with one to three N-terminal HMA domains. Phylogenetic analysis classified them into two subfamilies (Zn/Co/Cd/Pb and Cu/Ag P1B-ATPases). The genes are unevenly distributed across five chromosomes, with two segmental duplication events and an independent genome duplication event specific to dicots, and their promoters harbor abundant stress-responsive elements. Expression profiling under copper and cadmium stresses showed distinct tissue specificity: cadmium stress upregulated most genes in roots but suppressed them in leaves, whereas copper stress upregulated all genes in leaves and half of the genes in roots. This study represents the first systematic analysis of the SrHMA gene family, revealing its potential involvement in heavy-metal stress responses and providing candidate genes for future functional characterization and breeding of S. rebaudiana varieties with enhanced metal tolerance.

Neurodegenerative diseases share features of neuronal loss, neuroinflammation, and protein aggregation. The cGAS-STING pathway, a key DNA sensor, mediates neuroinflammation via TBK1-IRF3 and IKK-NF-κB axes, inducing type I interferons and pro-inflammatory cytokines. This pathway upregulates ZBP1, promotes PANoptosome assembly, and triggers PANoptosis, releasing DAMPs and creating a self-amplifying "inflammation-death" cycle. In Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, pathological proteins (Aβ, Tau, α-synuclein, TDP-43) or genetic defects (e.g., C9orf72 repeats) cause mitochondrial DNA leakage or genomic instability, activating this axis. This review highlights the cGAS-STING-PANoptotic cascade as a shared pathogenic mechanism and discusses the current evidence and remaining challenges in confirming this hypothesis.