BACKGROUND: Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet β cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed.
METHODS: db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays.
RESULTS: In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers.
CONCLUSIONS: PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A\'s suppressive effects on hepatic gluconeogenesis and lipogenesis.

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Introducing the exploration of stimulated CD4+ cells adenosine triphosphate (sATPCD4) levels for immune monitoring post non-small cell lung cancer (NSCLC) chemotherapy, the present study aimed to investigate its efficacy in gauging the potential risk of disease progression (PD) in patients with NSCLC. Therefore, a total of 89 patients with advanced NSCLC, who underwent chemotherapy between August 15 2022 and August 30 2023 at the Fifth Affiliated Hospital of Guangzhou Medical University (Guangzhou, China), were retrospectively studied. Patients were divided into the PD (n=21) and disease stability (non-PD; n=68) groups and their clinical data were compared. The thresholds for predicting PD were identified using receiver operating characteristics (ROC) curves. Multivariate logistic regression analysis was carried out to assess the association between peripheral blood markers and the incidence of PD. Therefore, post-chemotherapy, significant differences in white blood cell count, non-stimulated CD4+ cells ATP and sATPCD4 levels were obtained between patients in the PD and non-PD groups (P<0.05). In addition, sATPCD4 levels were notably decreased in the PD group compared with the non-PD group. Furthermore, ROC analysis revealed that the predictive threshold for PD was 224.5 ng/ml [area under the curve=0.887; 95% confidence interval, 0.811-0.963]. Additionally, patients with low immunity (ATP <224.5 ng/ml) exhibited a higher risk of PD compared with the high-immunity group (ATP >224.5 ng/ml; P<0.0001). Finally, multivariate logistic regression analysis suggested that sATPCD4 could serve as an independent factor for predicting NSCLC progression. Overall, the current study predicted that immune function could be possibly associated with the risk of PD in patients with NSCLC.

We present the design, fabrication, and testing of a low-cost, miniaturized detection system that utilizes chemiluminescence to measure the presence of adenosine triphosphate (ATP), the energy unit in biological systems, in water samples. The ATP-luciferin chemiluminescent solution was faced to a silicon photomultiplier (SiPM) for highly sensitive real-time detection. This system can detect ATP concentrations as low as 0.2 nM, with a sensitivity of 79.5 A/M. Additionally, it offers rapid response times and can measure the characteristic time required for reactant diffusion and mixing within the reaction volume, determined to be 0.3 ± 0.1 s. This corresponds to a diffusion velocity of approximately 44 ± 14 mm2/s.

P2X7 receptor activation by extracellular adenosine triphosphate (eATP) modulates different intracellular pathways, including pro-inflammatory and tumor-promoting cascades. ATP is released by cells and necrotic tissues during stressful conditions and accumulates mainly in the inflammatory and tumoral microenvironments. As a consequence, both the P2X7 blockade and agonism have been proposed as therapeutic strategies in phlogosis and cancer. Nevertheless, most studies have been carried out on the WT fully functional receptor variant. In recent years, the discovery of P2X7 variants derived by alternative splicing mechanisms or single-nucleotide substitutions gave rise to the investigation of these new P2X7 variants\' roles in different processes and diseases. Here, we provide an overview of the literature covering the function of human P2X7 splice variants and polymorphisms in diverse pathophysiological contexts, paying particular attention to their role in oncological and neuroinflammatory conditions.

Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug\'s ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 μM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

The Davydov model was conjectured to describe how an amide I excitation created during ATP hydrolysis in myosin might be significant in providing energy to drive myosin\'s chemomechanical cycle. The free energy surfaces of the myosin relay helix peptide dissolved in 2,2,2-trifluoroethanol (TFE), determined by metadynamics simulations, demonstrate local minima differing in free energy by only ~2 kT, corresponding to broken and stabilized hydrogen bonds, respectively. Experimental pump-probe and 2D infrared spectroscopy were performed on the peptide dissolved in TFE. The relative heights of two peaks seen in the pump-probe data and the corresponding relative volumes of diagonal peaks seen in the 2D-IR spectra at time delays between 0.5 ps and 1 ps differ noticeably from what is seen at earlier or later time delays or in the linear spectrum, indicating that a vibrational excitation may influence the conformational state of this helix. Thus, it is possible that the presence of an amide I excitation may be a direct factor in the conformational state taken on by the myosin relay helix following ATP hydrolysis in myosin.

Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca2+-ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins.

The complex architecture and biochemistry of the inner mitochondrial membrane generate ultra-structures with different phospholipid and protein compositions, shapes, characteristics, and functions. The crista junction (CJ) serves as an important barrier separating the cristae (CM) and inner boundary membranes (IBM). Thereby CJ regulates the movement of ions and ensures distinct electrical potentials across the cristae (ΔΨC) and inner boundary (ΔΨIBM) membranes. We have developed a robust and flexible approach to visualize the CJ permeability with super-resolution microscopy as a readout of local mitochondrial membrane potential (ΔΨmito) fluctuations. This method involves analyzing the distribution of TMRM fluorescence intensity in a model that is restricted to the mitochondrial geometry. We show that mitochondrial Ca2+ elevation hyperpolarizes the CM most likely caused by Ca2+ sensitive increase of mitochondrial tricarboxylic acid cycle (TCA) and subsequent oxidative phosphorylation (OXPHOS) activity in the cristae. Dynamic multi-parameter correlation measurements of spatial mitochondrial membrane potential gradients, ATP levels, and mitochondrial morphometrics revealed a CJ-based membrane potential overflow valve mechanism protecting the mitochondrial integrity during excessive cristae hyperpolarization.

Acute brain slices represent a workhorse model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue damage, it is unclear how microglia, the main immune cells and damage sensors of the CNS react to this injury and shape neuronal activity ex vivo. To this end, we investigated microglial phenotypes and contribution to network organization and functioning in acute brain slices. We reveal time-dependent microglial phenotype changes influenced by complex extracellular ATP dynamics through P2Y12R and CX3CR1 signalling, which is sustained for hours in ex vivo mouse brain slices. Downregulation of P2Y12R and changes of microglia-neuron interactions occur in line with alterations in the number of excitatory and inhibitory synapses over time. Importantly, functional microglia modulate synapse sprouting, while microglial dysfunction results in markedly impaired ripple activity both ex vivo and in vivo. Collectively, our data suggest that microglia are modulators of complex neuronal networks with important roles to maintain neuronal network integrity and activity. We suggest that slice preparation can be used to model time-dependent changes of microglia-neuron interactions to reveal how microglia shape neuronal circuits in physiological and pathological conditions.