Peripheral blood mononuclear cells (PBMCs) are commonly cryopreserved for immune cell profiling analyses, like flow cytometry. Cryopreservation of PBMCs may affect the recovery of immune cell subsets or the presence of cell-surface molecules, potentially skewing results compared to freshly isolated PBMCs. The ectonucleotidase CD39 promotes immunosuppression through the hydrolysis of extracellular nucleotides such as adenosine 5'-triphosphate (ATP) to limit P2X7 activation and downstream immune responses. Because of this, CD39 and P2X7 are emerging as potential biomarkers in cancer and other diseases. Using flow cytometry, this study demonstrated that an anti-human CD39 monoclonal antibody (mAb) (clone TU66) bound human embryonic kidney (HEK) 293 cells transfected with human CD39, but not mock-transfected HEK293 cells. In contrast, an anti-human P2X7 mAb (clone 1058613) bound similarly to HEK293 cells transfected with human P2X7 and non-transfected HEK293 cells. Using flow cytometry, this study then assessed the impact of cryopreservation on the population frequencies of immune cell subsets including CD39+ cells in human PBMC samples. These analyses revealed that cryopreservation had a minor, yet statistically significant impact on the population frequencies of CD4+, CD8+ and CD4-CD8- T cells. Cryopreservation also had a significant impact on the frequencies of CD39+CD19+ B cells and CD39+ non-classical monocytes. Overall, this study informs researchers and biobank workers about the need to consider the impact of cryopreservation when interpreting data from stored samples.

RNA synthesis by eukaryotic polymerases requires existing polynucleotides to serve as templates or primers. Here, we describe primer- and template-free RNA generation by human terminal nucleotidyltransferase 4B (TENT4B) via de novo polymerization of free nucleotides. We observed that recombinant TENT4B (rTENT4B) consumes ATP to yield inorganic pyrophosphate in the absence of a primer or template, concurrent with the appearance of oligomeric poly-adenosine RNA products. Remarkably, 5' labels on γ-phosphate-modified ATP or GTP are retained during polymerization in the presence of unlabeled nucleotide triphosphates (NTPs). These polymers are created at a similar efficiency irrespective of the inclusion of a primer, indicating robust RNA synthesis by rTENT4B from free NTPs. While canonical purine NTPs are favored, nucleotide diphosphates (NDPs) can also serve as substrates for rTENT4B-mediated de novo RNA polymerization. rTENT4B-mediated RNA synthesis using free adenosine nucleotides shows high processivity to generate 1000s-mers, whereas guanosine nucleotide polymerization is strongly and uniformly self-limited and yields a 3'-exonuclease-resistant oligonucleotide. Interrogation of other RNA polymerases reveals potential capacity for de novo polymerization using free ATP, albeit at significantly higher substrate concentrations and lower efficiency compared with rTENT4B. Our data provide definitive evidence of efficient template-free de novo RNA synthesis by a eukaryotic polymerase.

Posttranscriptional RNA modifications and their impact on the structure and function have been extensively studied on linear RNAs. As RNA modifications affect RNA structure, stability, localization, and translation, several methods have been developed to study modifications on linear RNAs. These modifications on RNAs ultimately regulate gene expression. One of the poorly characterized classes of RNA critical for gene expression is circular RNAs (circRNAs), which are ubiquitously expressed and are covalently closed RNA molecules. Although advances in sequencing technologies have greatly enhanced the identification of RNA modifications and their importance in gene regulation, the effects of these modifications on circRNA structure and function are only now emerging. Only a few modifications, like N[6]-methyladenosine (m[6]A), 5-methylcytosine (m[5]C), adenosine-to-inosine (A-to-I), and to a lesser extent N1-methyladenosine (m[1]A), have been reported so far for circRNAs. In this article, we discuss the importance of these modifications and their implications in circRNA function. Moreover, we will discuss molecular and computational methods for detecting circRNA modifications, to help researchers studying circRNAs select the most effective methods for investigating modifications in this novel class of circRNAs.

Pancreatic ductal adenocarcinoma (PDAC) remains refractory to immunotherapy, frequently characterized by an immune-excluded tumor microenvironment (TME). Oncogenic KRAS mutation, the predominant genetic driver in PDAC, serves as a master upstream regulator linking tumor-intrinsic signaling with microenvironmental remodeling. Through reshaping cytokine secretion and metabolic programs, mutant KRAS coordinates inflammatory and metabolic networks within the TME. Specifically, KRAS-linked cytokine networks, including tumor-cell-intrinsic mediators such as GM-CSF and microenvironment-associated inflammatory signals involving CXCLs, IL-6, TGF-β, and IL-1β, interact reciprocally with metabolic alterations, such as glycolytic activation, lactate accumulation, hypoxia adaptation, adenosine signaling, and altered amino acid and lipid metabolism. These pathways converge on shared regulatory nodes, notably STAT3, NF-κB, HIF-1α, and MYC, to drive immunosuppressive myeloid cell accumulation, stromal cell activation, extracellular matrix deposition, and restricted CD8[+] T-cell infiltration. Furthermore, lactate, adenosine, and physical stromal barriers reinforce these immunosuppressive cascades, thereby establishing spatial immune exclusion. This review summarizes recent evidence on KRAS-driven cytokine-metabolic crosstalk in PDAC and discusses how this genotype-associated regulatory pattern governs immune exclusion, offering novel insights into overcoming immunotherapy resistance.

m[6]A modification has been shown to play a role in regulating HBV RNA expression. However, it remains unclear whether the m[6]A levels of HBV pregenomic RNA (pgRNA) are related to HBV replication and liver function. Serum HBV pgRNA-m[6]A levels were determined by T3 DNA ligase assay. HBV DNA copy numbers and gene expression were detected by TaqMan assay and RT-qPCR, respectively. Liver function was evaluated using clinical laboratory indicators. The TET-off stable HBV-producing cell line HepAD38 and targeted pgRNA demethylation by SunTag system (TRADES) were used for the site-specific m[6]A editing. Flow cytometry was used to measure the cytokine levels in the supernatant of cell culture. Serum HBV pgRNA-m[6]A levels were positively correlated with HBV DNA copy number, pgRNA expression and liver function indicators (all p < 0.05). Both pgRNA-m[6]A levels and pgRNA expression were significantly increased in patients with high virus load (HBV DNA > 1.0 × 10[7] IU/mL). Site-specific demethylation of pgRNA1907-m[6]A significantly reduced HBV DNA, hepatitis B e antigen (HBeAg) and HBV pgRNA, while induced levels of interferon alfa 2 (IFN-α2) and apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A). pgRNA-m[6]A modification of HBV associates with viral replication and liver function indicators. Site-specific m[6]A demethylation of pgRNA provides novel insight into anti-HBV treatment.

This study investigated the applicability of a handheld electrical impedance meter (EIM) for nondestructive evaluation of the freshness of broiler breast meat during refrigerated storage. Impedance ratios (2/100 kHz) were measured on Days 0, 1, 2, 3, 5, 7, and 10 of refrigerated storage and were compared with the K-values obtained through high-performance liquid chromatography (HPLC) analysis of adenosine triphosphate (ATP) degradation products. The impedance ratio decreased consistently over time, with SD decreasing from 0.36 on Day 0 to 0.07 on Day 10. A quadratic regression model showed that the impedance ratio decreased with the number of storage days (R[2] = 0.7561). The K-value increased over time and was also well described by a quadratic regression model (R[2] = 0.8395). The impedance ratio was strongly negatively correlated with the K-value (r = -0.730; n = 140; p < 0.001), supporting its utility for tracking freshness-related changes. Although the K-value demonstrated a slightly higher explanatory power, the impedance ratio showed sufficient accuracy for practical applications. These findings suggest that the EIM method offers a rapid, simple, and nondestructive alternative to conventional biochemical assays, with potential for implementation in poultry processing and retail environments for on-site freshness assessment.

Epigenetic editing, particularly N[6]-methyladenosine (m[6]A) modification, represents a promising therapeutic strategy by silencing genes without altering DNA sequence. However, in vivo epigenetic intervention of neuroinflammation remains challenging and has rarely been explored. Here we developed a hybrid epigenetic nanomodulator, siMETTL3-hNVs, by integrating natural microglia-derived nanovesicles (NVs) with synthetic liposomes pre-loading small interfering RNA targeting the m[6]A writer methyltransferase-like 3 (METTL3). Natural NVs enabled siMETTL3-hNVs to achieve inflamed-brain delivery through CCR2-CCL2 chemotaxis and caveolae-mediated transcytosis across the blood-brain barrier. More importantly, relying on abundant cytokine receptors on the NVs, siMETTL3-hNVs served as decoys to neutralize pro-inflammatory cytokines, synergizing with the intracellular silencing of METTL3 to drive microglial M2 repolarization. In female mouse models of acute neuroinflammation and radiation-induced brain injury, siMETTL3-hNVs treatment significantly reduced cytokine levels, attenuated hippocampal damage, and ameliorated cognitive deficits. This work overcomes critical delivery bottlenecks in m[6]A-based therapeutics and establishes a robust strategy for epigenetic reprogramming of neuroinflammation.

Early-life nutrition may influence lifelong ageing, yet human evidence is scarce. Using Britain's postwar sugar rationing as a natural experiment, we examine its long-term effects in 64,809 United Kingdom Biobank participants. Exposure to sugar rationing during the first 1,000 days of life is associated with a 9% lower incidence of hallmark-related disease, with a hazard ratio of 0.91 and a 95% confidence interval of 0.88-0.94, and a 19% lower risk of all-cause mortality, with a hazard ratio of 0.81 and a 95% confidence interval of 0.69-0.93. Mediation analysis indicates that the survival association is statistically mediated, by approximately 60%, through differences in incident hallmark-related disease. Rationed individuals show 1.0-1.2-year younger biological ages across multiple clocks and lower organ ages, particularly in the lung, heart, and liver. Proteomic profiling identifies 47 altered proteins, with enrichment of adenosine monophosphate-activated protein kinase and longevity pathways and suppression of mechanistic target of rapamycin signaling. These findings are consistent with international recommendations to limit free or added sugars from the World Health Organization, United States Dietary Guidelines, and American Heart Association, and may inform policy discussions related to sugar taxation and infant food and marketing policies under the United Nations 2030 Agenda.

Visualization of adenosine triphosphate (ATP) dynamics in living cells is crucial for revealing metabolic abnormalities in tumors. Although DNA-based fluorescent probes show promise for biomolecular detection, their application in intracellular imaging faces several challenges, including insufficient cellular uptake efficiency and nonspecific signal activation prior to reaching the cellular target. To address these limitations, we constructed a tumor-targeted, aptamer-based, photocleavable DNA nanoflower (TAPE) system via rolling circle amplification (RCA). The TAPE integrates three core functional modules: the AS1411 aptamer module for tumor cell targeting via nucleolin binding, a cholesterol-modified module for enhanced membrane interaction and delivery, and a photocontrollable ATP-sensing module. In vitro characterization confirmed that TAPE's dual-input (UV light and ATP) sensing logic requires prior photocleavage followed by ATP binding to trigger fluorescence signals. The system demonstrated an ATP detection limit of 39.18 μM with high selectivity over other nucleotide triphosphates. Cellular uptake studies showed that TAPE was efficiently internalized by nucleolin-positive A549 cancer cells in an AS1411 aptamer-dependent manner, with a positive uptake rate of 98.5%. This uptake efficiency was significantly higher than that in control cells (P < 0.0001). In cellular experiments, TAPE enabled the spatiotemporally controlled imaging of intracellular ATP, successfully monitoring ATP level fluctuations induced by pharmacological agents etoposide under photocleavage-mediated activation. In conclusion, this study develops a spatiotemporally controlled intracellular ATP imaging strategy, offering a reliable platform for tumor research.