Search

Simple Summary: Cutaneous T-cell lymphoma (CTCL) is a rare type of T-lymphocyte malignancy strongly calling for novel therapies. Virotherapy by means of oncolytic viruses that are able to kill cancer cells, while sparing healthy cells, is a promising innovative form of anticancer immunotherapy. The aim of this study was to investigate the potential of an oncolytic parvovirus, H-1PV, to induce selective killing (oncolysis) of CTCL cells and suppress the growth of CTCL spheroids. We demonstrated that H-1PV treatment led to oncolysis in tumor, but not in control normal cells. Oncolysis ensued despite pro-survival protein overexpression and was associated with the release of danger-signaling molecules. In heterotypic CTCL spheroids, H-1PV induced spheroid growth suppression and, upon co-culturing with peripheral blood mononuclear cells, spheroid infiltration with immune cells. In summary, we gathered the first preclinical data showing that H-1PV holds significant potential to become a novel viroimmunotherapeutic agent against CTCL. The rat protoparvovirus H-1 (H-1PV) is an oncolytic virus known for its anticancer properties in laboratory models of various human tumors, including non-Hodgkin lymphomas (NHL) of B-cell origin. However, H-1PV therapeutic potential against hematological malignancies of T-cell origin remains underexplored. The aim of the present study was to conduct a pilot preclinical investigation of H-1PV-mediated oncolytic effects in cutaneous T-cell lymphoma (CTCL), a type of NHL that is urgently calling for innovative therapies. We demonstrated H-1PV productive infection and induction of oncolysis in both classically grown CTCL suspension cultures and in a novel, in vivo-relevant, heterotypic spheroid model, but not in healthy donor controls, including peripheral blood mononuclear cells (PBMCs). H-1PV-mediated oncolysis of CTCL cells was not prevented by Bcl-2 overexpression and was accompanied by increased extracellular ATP release. In CTCL spheroid co-cultures with PBMCs, increased spheroid infiltration with immune cells was detected upon co-culture treatment with the virus. In conclusion, our preclinical data show that H-1PV may hold significant potential as an ingenious viroimmunotherapeutic drug candidate against CTCL. [ABSTRACT FROM AUTHOR]

Objective: We aimed to investigate antiplatelet drug resistance utilizing light transmission-lumiaggregometry (LT-LA) and the Platelet Function Analyzer-100 (PFA-100) in patients undergoing cardiovascular surgery. Materials and Methods: The study included 60 patients diagnosed with stable coronary artery disease and peripheral vascular diseases that required surgery. Participants were divided into three groups: patients receiving aspirin (ASA) (n=21), patients receiving clopidogrel (CLO) (n=19), and patients receiving dual therapy (ASA+CLO) (n=20). Aggregation and secretion tests by LT-LA and closure time by the PFA-100 were used to measure antiplatelet drug resistance. Results: Based on the adenosine diphosphate (ADP)-induced aggregation test, 43% of patients were resistant to ASA, 22% to CLO, and 15% to dual therapy. Diabetes, hypertension, and hyperlipidemia were the most commonly identified comorbid disorders. In patients with comorbid risk factors, the median value of platelet aggregation response to ADP was significantly higher in the ASA group than in the CLO and dual therapy groups (p=0.0001). In patients receiving ASA monotherapy, the maximum amplitude of aggregation response to platelet agonists was ≥70% in 43% of patients for ADP and 28% for collagen by LT-LA. Elevated ADP (≥0.29 nmol) and collagen (≥0.41 nmol)-induced adenosine triphosphate release were found by LT-LA in 66% of patients utilizing an ADP agonist and 80% of patients using a collagen agonist undergoing ASA therapy. Closure times obtained with the PFA-100 were normal in 28% of patients using collagen-ADP cartridges and 62% of patients using collagen-epinephrine (CEPI) cartridges who received ASA. Recurrent thrombosis and bleeding were observed in 12 (20%) patients with cardiovascular disease. Three of these individuals (25%) showed ASA resistance with normal responses to ADP-induced aggregation (≥70%) and secretion (≥0.29 nmol), as well as normal CEPI closure times. Conclusion: Our findings suggest that antiplatelet drug monitoring by LT-LA and PFA-100 may be useful for high-risk and complicated cardiovascular patients. [ABSTRACT FROM AUTHOR]

Cisplatin is a common chemotherapeutic drug for treating ovarian cancer, but its clinical efficacy is hampered by intrinsic and acquired resistance. Previous studies had shown inhibiting oxidative phosphorylation overcomes cisplatin resistance in ovarian cancer. Studies reveal that bedaquiline, a clinically available antimicrobial drug, inhibits cancer via targeting mitochondria. This study systematically assessed the efficacy of bedaquiline in ovarian cancer and its underlying mechanism. Using a panel of ovarian cancer cell lines and normal ovary cells, we demonstrated bedaquiline is selective for anti-ovarian cancer activities. Furthermore, the sensitivity varied among different ovarian cancer cell lines regardless of their sensitivity to cisplatin. Bedaquiline inhibited growth, survival and migration, through decreasing levels of ATP synthase subunit, complex V activity, mitochondrial respiration and ATP. We further found that ovarian cancer displayed increased levels of ATP, oxygen consumption rate (OCR), complex V activity and ATP synthase subunits compared to normal counterpart. Combination index analysis showed that bedaquiline and cisplatin is synergistic. Bedaquiline remarkably enhanced the efficacy of cisplatin in inhibiting ovarian cancer growth in mice. Our study provides evidence to repurpose bedaquiline for ovarian cancer treatment and suggests that ATP synthase is a selective target to overcome cisplatin resistance in ovarian cancer. [ABSTRACT FROM AUTHOR]

The mitochondria have an important role in modulating cell cycle progression, cell survival, and apoptosis. In the adult heart, the cardiac mitochondria have a unique spatial arrangement and occupy nearly one-third the volume of a cardiomyocyte, being highly efficient for converting the products of glucose or fatty acid metabolism into adenosine triphosphate (ATP). In cardiomyocytes, the decline of mitochondrial function reduces ATP generation and increases the production of reactive oxygen species, which generates impaired heart function. This is because mitochondria play a key role in maintaining cytosolic calcium concentration and modulation of muscle contraction, as ATP is required to dissociate actin from myosin. Beyond that, mitochondria have a significant role in cardiomyocyte apoptosis because it is evident that patients who have cardiovascular diseases (CVDs) have increased mitochondrial DNA damage to the heart and aorta. Many studies have shown that natural products have mitochondria-modulating effects in cardiac diseases, determining them as potential candidates for new medicines. This review outlines the leading plant secondary metabolites and natural compounds derived from microorganisms as modulators of mitochondrial dysfunctions associated with CVDs. [ABSTRACT FROM AUTHOR]

Background: Phosphorus cardiovascular magnetic resonance spectroscopy (31P-CMRS) has emerged as an important tool for the preclinical assessment of myocardial energetics in vivo. However, the high rate and diminutive size of the mouse heart is a challenge, resulting in low resolution and poor signal-to-noise. Here we describe a refined high-resolution 31P-CMRS technique and apply it to a novel double transgenic mouse (dTg) with elevated myocardial creatine and creatine kinase (CK) activity. We hypothesised a synergistic effect to augment energetic status, evidenced by an increase in the ratio of phosphocreatine-to-adenosine-triphosphate (PCr/ATP). Methods and results: Single transgenic Creatine Transporter overexpressing (CrT-OE, n = 7) and dTg mice (CrT-OE and CK, n = 6) mice were anaesthetised with isoflurane to acquire 31P-CMRS measurements of the left ventricle (LV) utilising a two-dimensional (2D), threefold under-sampled density-weighted chemical shift imaging (2D-CSI) sequence, which provided high-resolution data with nominal voxel size of 8.5 µl within 70 min. (1H-) cine-CMR data for cardiac function assessment were obtained in the same imaging session. Under a separate examination, mice received invasive haemodynamic assessment, after which tissue was collected for biochemical analysis. Myocardial creatine levels were elevated in all mouse hearts, but only dTg exhibited significantly elevated CK activity, resulting in a 51% higher PCr/ATP ratio in heart (3.01 ± 0.96 vs. 2.04 ± 0.57—mean ± SD; dTg vs. CrT-OE), that was absent from adjacent skeletal muscle. No significant differences were observed for any parameters of LV structure and function, confirming that augmentation of CK activity does not have unforeseen consequences for the heart. Conclusions: We have developed an improved 31P-CMRS methodology for the in vivo assessment of energetics in the murine heart which enabled high-resolution imaging within acceptable scan times. Mice over-expressing both creatine and CK in the heart exhibited a synergistic elevation in PCr/ATP that can now be tested for therapeutic potential in models of chronic heart failure. [ABSTRACT FROM AUTHOR]

Background: Our research is designed to explore the role of 5-FU and Pulsatilla decoction (PD) through modulation of Immunogenic cell death (ICD) for the co-treatment of Colorectal cancer (CRC). Materials and Methods: Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assays. Cell apoptosis was assessed using flow cytometry. Phosphorylation of STAT3 and expression of Mcl-1 and Bcl-xl were measured by Western blot assays. The levels of ATP and HMGB1 in the supernatants of the culture medium were analyzed by ATP assays and the HMGB1 enzyme linked immunosorbent assay kit. The cell surface levels of CRT were measured by immunofluorescence assays. The tumor growth was analyzed in mice. Results: PD increased 5-FU-induced ICD in CRC cells, as demonstrated by the extracellular levels of adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1), and the surface levels of calreticulin (CRT). Our mechanism study showed that PD promoted 5-FU-induced ICD by inactivating signal transducer and activator of transcription 3 (STAT3). Furthermore, the co-treatment of 5-FU and PD further promoted 5-FU-induced CRT expression and T cell infiltration in vivo. Tumorigenicity analysis revealed that 5-FU combined with PD notably reduced tumor growth. Conclusion: This study indicated that PD enhances 5-FU-induced ICD and anti-tumor effect in CRC by inactivating STAT3. The combined application of 5-FU with PD may improve the anti-tumor activity of 5-FU in CRC. [ABSTRACT FROM AUTHOR]

The brain is an organ that consumes a substantial amount of oxygen, and a reduction in oxygen concentration can rapidly lead to significant and irreversible brain injury. The progression of brain injury during hypoxia involves the depletion of intracellular adenosine triphosphate (ATP) due to decreased oxidative phosphorylation in the inner mitochondrial membrane. Allopurinol is a purine analog inhibitor of xanthine oxidoreductase that protects against hypoxic/ischemic brain injury; however, its underlying mechanism of action remains unclear. In addition, febuxostat is a non-purine xanthine oxidoreductase inhibitor with a different inhibitory mechanism from allopurinol. The impact of febuxostat on brain injury has not been well investigated. Therefore, this study aimed to examine brain ATP and its catabolite levels in the presence or absence of allopurinol and febuxostat under hypoxic conditions by inactivating brain metabolism using focal microwave irradiation. The hypoxic treatment caused a decrease in the adenylate energy charge and ATP levels and an increase in its catabolic products in mouse brains. The febuxostat group showed higher energy charge and ATP levels and lower ATP catabolites than the control group. Notably, despite the comparable suppression of uric acid production in both inhibitor groups, allopurinol treatment was less effective than febuxostat. These results suggest that febuxostat effectively prevents hypoxia-induced ATP degradation in the brain and that its effect is more potent than allopurinol. This study will contribute to developing therapies for improving hypoxia-induced brain dysfunction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

The methanogenic archaeon Methanosarcina acetivorans has one of the largest known archaeal genomes. With 53 histidine kinases (HK), it also has the largest set of signal transduction systems. To gain insight into the hitherto not very well understood signal transduction in Archaea and M. acetivorans in particular, we have categorised the predicted HK into four types based on their H-box using an in silico analysis. Representatives of three types were recombinantly produced in Escherichia coli and purified by affinity chromatography. All investigated kinases showed ATP binding and hydrolysis. The MA_type 2 kinase, which lacks the classical H-box, showed no autokinase activity. Furthermore, we could show that M. acetivorans possesses an above-average number of response regulators (RR), consisting of only a REC domain (REC-only). Using the hybrid kinase MA4377 as an example we show that both intra-and intermolecular transphosphorylation to REC domains occur. These experiments are furthermore indicative of complex phosphorelay systems in M. acetivorans and suggest that REC-only proteins act as a central hub in signal transduction in M. acetivorans., (© 2025 The Author(s). Environmental Microbiology published by John Wiley & Sons Ltd.)

Capsaicin, a polyphenol, is known to regulate energy expenditure and thermogenesis in adipocytes and muscles. However, its role in modulating uncoupling proteins (UCPs) and adenosine triphosphate (ATP)-dependent thermogenesis in muscles remains unclear. This study investigated the mechanisms underlying the role of capsaicin in modulating the UCP- and ATP-dependent thermogenesis in C2C12 myoblasts, as well as the gastrocnemius (GM) and soleus muscles (SM) of mice. We employed molecular dynamics (MD), quantitative real-time polymerase chain reactions (qRT-PCR), immunoblots, staining methods, and assay kits to investigate the role of capsaicin on thermogenesis and its modulatory roles on the transient receptor potential cation channel subfamily V member 1 (TRPV1) and α-/β-adrenergic receptors (ARs) using in vitro and in vivo models. Our findings demonstrate that capsaicin treatment in high-fat diet-induced obese mice reduces weight gain and elevates the expression of UCP- and ATP-dependent thermogenic effectors through ATP-consuming calcium and creatine futile cycles. In vitro and in vivo models capsaicin treatment elevated the expression of sarcoendoplasmic/endoplasmic reticulum calcium ATPases (SERCA-1 and -2), ryanodine receptors (RYR-1 and -2), uncoupling proteins (UCP-2 and -3), creatine kinase B (CKB), and creatine kinase mitochondrial 2 (CKMT2), through activation of TRPV1, α1-, β2-, and β3-AR as well as the suppressed expression of α2-AR. Furthermore, our results also indicate that capsaicin promotes myotube development and enhances lipid metabolism in C2C12 cells. We found that capsaicin increased intracellular Ca 2+ levels and the expression of the voltage-dependent anion channel (VDAC) and mitochondrial calcium uniporter (MCU), suggesting that elevated mitochondrial Ca 2+ levels boost the expression of oxidative phosphorylation protein complexes via the activation of the ATP-futile cycle. Mechanistic studies in C2C12 cells revealed that TRPV1 is likely dispensable for capsaicin-induced thermogenesis, and TRPV1 and α1-AR may synergistically induce thermogenesis. Collectively, our findings have uncovered a novel mechanism of UCP- and ATP-dependent thermogenesis and its associated pathways in both cellular and animal models which is crucial for designing therapeutic strategies to address obesity and associated metabolic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Chopping and salting are two important processing steps in emulsified meat products. Effects of chopping and salting on metabolic process, protein functionality, and ultrastructure of pre-rigor silver carp muscle, and how these three aspects changed during rigor transformation were explored. Chopping caused an accelerated loss of adenosine triphosphate (ATP) from 1.16 μmol/g to 0.16 μmol/g, and salt addition inhibited accumulation of hypoxanthine nucleoside (HxR) and hypoxanthine (Hx). Similarly, chopping led to faster decrease of glycogen from 4.59 mg/g to 1.50 mg/g and increase in lactic acid from 0.52 mmol/g protein to 0.82 mmol/g protein, and salt exerted an inhibition effect. In agreement with ATP and glycogen breakdown, metabolic profiling revealed that chopping and salting altered the metabolism in fatty acids and amino acids during rigor transformation. After rigor transformation, chopping with salt led to significant reduction in radical scavenging ability, accompanied by greater loss of sulfhydryl groups. Salt also promoted protein denaturation, evidenced by increased surface hydrophobicity and decreased intrinsic fluorescence. The ultrastructure of fish muscle after chopping or chopping with salt was similar between pre- and post-rigor stages. The abovementioned findings can provide valuable insight into the production of fish products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Kinesins are a diverse superfamily of microtubule-based motors that perform fundamental roles in intracellular transport, cytoskeletal dynamics and cell division. These motors share a characteristic motor domain that powers unidirectional motility and force generation along microtubules, and they possess unique tail domains that recruit accessory proteins and facilitate oligomerization, regulation and cargo recognition. The location, direction and timing of kinesin-driven processes are tightly regulated by various cofactors, adaptors, microtubule tracks and microtubule-associated proteins. This Review focuses on recent structural and functional studies that reveal how members of the kinesin superfamily use the energy of ATP hydrolysis to transport cargoes, depolymerize microtubules and regulate microtubule dynamics. I also survey how accessory proteins and post-translational modifications regulate the autoinhibition, cargo binding and motility of some of the best-studied kinesins. Despite much progress, the mechanism and regulation of kinesins are still emerging, and unresolved questions can now be tackled using newly developed approaches in biophysics and structural biology., Competing Interests: Competing interests: The author declares no competing interests., (© 2024. Springer Nature Limited.)

The Hsp70 chaperone protein system is an essential component of the protein folding and homeostasis machinery in E.Coli. Hsp70 is a three domain, 70 kDa protein which functions as an allosteric system cycling between an ADP-bound state where the three domains are loosely coupled via a flexible interdomain linker and an ATP-bound state where they are tightly coupled into a single entity. The structure-function model of this protein proposes an allosteric connection between the 45 kDa Nucleotide Binding Domain (NBD) and the 25 kDa Substrate Binding Domain (SBD) and Lid Domain which operates through the inter NBD-SBD linker. X-Ray crystallography and NMR spectroscopy have provided structures of the end states of the functional cycle of this protein, bound to ADP and ATP. We have used MD simulations to study the transitions between these end states and allosteric communication in this system. Our results largely validate the experimentally derived allosteric model of function, but shed additional light on the flow of allosteric information in the SBD + Lid. Specifically, we find that the Lid domain has a double-hinged structure with the potential for greater conformational flexibility than was hitherto expected.Communicated by Ramaswamy H. Sarma.

Anticancer effects of high-dose vitamin C (VC) have been evaluated on many cancer cell lines, and its efficacy in clinical trials and in combination with anticancer drugs or radiation have been reported; however, its effect on gastric cancer and its mechanisms remain unclear. In the present study, the cell growth inhibitory/lethal effects of high-dose ascorbic acid (AsA), a reduced form of VC was examined on three gastric cancer cell lines. Of these, signet ring cell carcinoma NUGC-4 cells were the most sensitive, but the effects were small and limited in normal cells. Second, high-dose AsA was effective in NUGC-4 cells, whereas dehydroascorbic acid, an oxidized form of VC, was less effective. Third, high-dose AsA showed stronger cell growth inhibitory/lethal effects on floating cells than on adherent cells, and was effective even under hypoxic microenvironment conditions. A single 1-h treatment of high-dose AsA strongly inhibited cell growth, causing apoptosis-like cell death over 72 h after treatment, triggered by hydrogen peroxide generation, actin abnormality, DNA synthesis suppression, DNA damage induction, and ATP level decrease. The effects of high-dose AsA were inhibited either by adding or chelating iron ions, but was not affected via inhibiting AsA transport. Inhibition of glutathione synthesis enhanced the anticancer effects of high-dose AsA. These results indicate that a single high-dose of AsA induces cancer cell-selective, sustained cell growth inhibition and cell death, and these effects may be regulated by iron ion and/or intracellular oxidative stress levels in human gastric signet-ring cell carcinoma-derived NUGC-4 cells., Competing Interests: Declaration of competing interest Yasukazu Saitoh reports equipment, drugs, or supplies was provided by JSPS KAKENHI. Yasukazu Saitoh reports writing assistance was provided by Prefectural University of Hiroshima Scientific Research Promotion Grant. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The cause and consequences of inosine monophosphate (IMP) formation when adenosine triphosphate (ATP) declines during muscular contractions in vivo are not fully understood. The purpose of this study was to examine the role of IMP formation in the maintenance of the Gibbs free energy for ATP hydrolysis (ΔG ATP ) during dynamic contractions of increasing workload and the implications of ATP loss in vivo. Eight males (median 27.5, 25-35 yr range) completed an 8-min incremental protocol [2-min stages of isotonic knee extensions (0.5 Hz)] in a 3-T magnetic resonance (MR) system. Phosphorus MR spectra were obtained from the knee extensor muscles at rest and during contractions and recovery. Although the ATP demand during contractions was met primarily by oxidative phosphorylation, [ATP] decreased from 8.2 mM to 7.5 (range 6.4-8.0) mM and [IMP] increased from 0 mM to 0.6 (0.1-1.7) mM. Modeling showed that, in the absence of IMP formation, excess adenosine diphosphate (ADP) would result in a less favorable ΔG ATP ( P < 0.001). Neither [ATP] nor [IMP] had returned to baseline following 10 min of recovery ( P < 0.001). Notably, Δ[ATP] was linearly related to the post-contraction reduction in muscle oxidative capacity ( r = 0.74, P = 0.037). Our results highlight the importance of IMP formation in preserving cellular energy status by avoiding increases in ADP above that necessary to stimulate energy production pathways. However, the consequence of IMP formation was an incomplete recovery of [ATP], which in turn was related to decreased muscle oxidative capacity following contractions. These results likely have implications for the capacity to generate adequate energy during repeated bouts of muscular work. NEW & NOTEWORTHY An ∼9% decline in [ATP] led to the formation of inosine monophosphate (IMP) during submaximal muscular contractions. Modeling revealed IMP formed to preserve a favorable energy state (ΔG ATP ) by minimizing large increases in [ADP], whereas the loss of [ATP] did not alter ΔG ATP . [ATP] did not recover by 10 min, and the loss of [ATP] was associated with a reduced oxidative capacity, providing a new link between [ATP] loss and an impaired energetic capacity in vivo.

Neurodegenerative diseases typically emerge after an extended prodromal period, underscoring the critical importance of initiating interventions during the early stages of brain aging to enhance later resilience. Changes in presynaptic active zone proteins ("PreScale") are considered a dynamic, resilience-enhancing form of plasticity in the process of early, still reversible aging of the Drosophila brain. Aging, however, triggers significant changes not only of synapses but also mitochondria. While the two organelles are spaced in close proximity, likely reflecting a direct functional coupling in regard to ATP and Ca 2+ homeostasis, the exact modes of coupling in the aging process remain to understood. We here show that genetic manipulations of mitochondrial functional status, which alters brain oxidative phosphorylation, ATP levels, or the production of reactive oxygen species (ROS), can bidirectionally regulate PreScale during early Drosophila brain aging. Conversely, genetic mimicry of PreScale resulted in decreased oxidative phosphorylation and ATP production, potentially due to reduced mitochondrial calcium (Ca 2+ ) import. Our findings indicate the existence of a positive feedback loop where mitochondrial functional state and PreScale are reciprocally coupled to optimize protection during the early stages of brain aging., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests. This manuscript is an original contribution not previously published and not under consideration for publication elsewhere., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Although various studies have been conducted, the detailed mechanisms of food allergy remain a topic of ongoing debate. Recently, researchers have reported that extracellular adenosine triphosphate (eATP), a member of damage-associated molecular patterns secreted by stressed cells, plays a critical role in the progression of asthma and atopic dermatitis. These studies suggest that dysregulated eATP significantly influences various aspects of disease progression, from bodily sensitization to the emergence of clinical manifestations. Given the shared pathogenic mechanisms among asthma, atopic dermatitis, and food allergies, we hypothesize that eATP may also serve as a crucial regulator in the development of food allergies. To elucidate this hypothesis, we first summarize the evidence and limitations of food allergy theories, then discuss the roles of eATP in allergic diseases. We conclude with speculative insights into the potential influence of eATP on food allergy development, aiming to inspire further investigation into the molecular mechanisms of food allergies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Purpose: Pannexin-1 (PANX1) channel participates in the development and progression of many tumor types, however, its role of PANX1 in invasive pituitary adenoma (PA) remains unknown. The current study was designed to investigate the role of PANX1 in invasion of PA., Methods: We examined the expression of PANX1 in 116 surgical invasion and non-invasion PA samples (60 for bulk transcriptome and 56 for immunohistochemistry). The effects of PANX1 on PA growth were assessed in vitro and xenograft models. Meanwhile, the metabolism changes of PA cells are explored via transcriptomics and metabolomics using integration strategy., Results: PANX1 is significantly upregulated in invasive PA compared with noninvasive PA and pituitary gland, and have a potential diagnostic signature for invasive PA. Accordingly, overexpression of PANX1 could promote the proliferation and invasion of GH3 and MMQ cell lines in vitro and in vivo. Further metabolomics results confirme that overexpression of PANX1 could trigger changes in several metabolic pathways of GH3 cells. Among the dysregulated cellular metabolites, decreased intracellular ATP suggeste that PANX1 may promote the invasion of PA through impacting extracellular ATP concentration. Mechanistically, extracellular ATP might promote Ca 2+ influx and upregulated the expression of MMP2/9 by activating P2X7R. Additionally, PANX1-ATP-P2 X7R signaling pathway might enhance GH3 cell invasion by remodeling the actin cytoskeleton., Conclusion: Our findings point to a pivotal role of PANX1 in promoting PA invasion, which indicated a potential therapeutic target for invasive PA., Competing Interests: Declarations. Ethics approval and consent to participate: This study complied with the Declaration of Helsinki and animal ethical statement, which was approved by the Ethics Committee of Xinqiao Hospital, Army Medical University (authorization ID: 2022-356-01 and AMUWEC20224532). Patients provided written informed consent to participate in the study. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Adenosine triphosphate plays a vital role in providing energy and enabling key cellular processes through interactions with binding proteins. The increasing amount of protein sequence data necessitates computational methods for identifying binding sites. However, experimental identification of adenosine triphosphate-binding residues remains challenging. To address the challenge, we developed a multi-window convolutional neural network architecture taking pre-trained protein language model embeddings as input features. In particular, multiple parallel convolutional layers scan for motifs localized to different window sizes. Max pooling extracts salient features concatenated across windows into a final multi-scale representation for residue-level classification. On benchmark datasets, our model achieves an area under the ROC curve of 0.95, significantly improving on prior sequence-based models and outperforming convolutional neural network baselines. This demonstrates the utility of pre-trained language models and multi-window convolutional neural networks for advanced sequence-based prediction of adenosine triphosphate-binding residues. Our approach provides a promising new direction for elucidating binding mechanisms and interactions from primary structure., Competing Interests: Declaration of competing interest I, The Le, hereby declare that I have no financial interests or relationships with any organizations that could potentially influence the subject matter of this work. I also confirm that I do not hold any professional or personal affiliations that may be perceived as affecting the impartiality and objectivity of my research. I have received no funding, grants, or honoraria related to the research presented in this work. Additionally, I have no personal relationships or collaborations that might pose a conflict of interest. This work is conducted with complete transparency, and I am committed to upholding the highest standards of integrity in my scholarly contributions., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Astrocytes are multi-functional glial cells in the central nervous system that play critical roles in modulation of metabolism, extracellular ion and neurotransmitter levels, and synaptic plasticity. Astrocyte-derived signaling molecules mediate many of these modulatory functions of astrocytes, including vesicular release of ATP. In the present study, we used a unique genetic mouse model to investigate the functional significance of astrocytic exocytosis of ATP. Using primary cultured astrocytes, we show that loss of vesicular nucleotide transporter (Vnut), a primary transporter responsible for loading cytosolic ATP into the secretory vesicles, dramatically reduces ATP loading into secretory lysosomes and ATP release, without any change in the molecular machinery of exocytosis or total intracellular ATP content. Deletion of astrocytic Vnut in adult mice leads to increased anxiety, depressive-like behaviors, and decreased motivation for reward, especially in females, without significant impact on food intake, systemic glucose metabolism, cognition, or sociability. These behavioral alterations are associated with significant decreases in the basal extracellular dopamine levels in the nucleus accumbens. Likewise, ex vivo brain slices from these mice show a strong trend toward a reduction in evoked dopamine release in the nucleus accumbens. Mechanistically, the reduced dopamine signaling we observed is likely due to an increased expression of monoamine oxidases. Together, these data demonstrate a key modulatory role of astrocytic exocytosis of ATP in anxiety, depressive-like behavior, and motivation for reward, by regulating the mesolimbic dopamine circuitry., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Toxic small alarmone synthetase (toxSAS) enzymes constitute a family of bacterial effectors present in toxin-antitoxin and secretion systems. toxSASs act through either translation inhibition mediated by pyrophosphorylation of transfer RNA (tRNA) CCA ends or synthesis of the toxic alarmone adenosine pentaphosphate ((pp)pApp) and adenosine triphosphate (ATP) depletion, exemplified by FaRel2 and FaRel, respectively. However, structural bases of toxSAS neutralization are missing. Here we show that the pseudo-Zn 2+ finger domain (pZFD) of the ATfaRel2 antitoxin precludes access of ATP to the pyrophosphate donor site of the FaRel2 toxin, without affecting recruitment of the tRNA pyrophosphate acceptor. By contrast, (pp)pApp-producing toxSASs are inhibited by Tis1 antitoxin domains though occlusion of the pyrophosphate acceptor-binding site. Consequently, the auxiliary pZFD of AT2faRel is dispensable for FaRel neutralization. Collectively, our study establishes the general principles of toxSAS inhibition by structured antitoxin domains, with the control strategy directly coupled to toxSAS substrate specificity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

In Leishmania, the nucleotide-sugar UDP-galactose can be synthesized by a salvage pathway, the Isselbacher route, involving phosphorylation of galactose and the action of UDP-sugar pyrophosphorylase. The first enzyme of the pathway, galactokinase, has yet to be studied in this parasite. Here, we report a molecular and biochemical characterization of this enzyme in Leishmania mexicana. We showed that recombinant galactokinase (LmxGALK) phosphorylates galactose in the presence of ATP with K m values of 0.077 mM for galactose and 0.017 mM for ATP. We proved by immunodetection that GALK is expressed in promastigotes and amastigotes of L. mexicana, L. braziliensis and L. infantum. In agreement with the presence of a type 1 peroxisome-targeting signal sequence present at the C-terminus of LmxGALK, the protein is localized mostly within glycosomes as shown by selective membrane permeabilization with digitonin, differential centrifugation, and immunofluorescence. Indeed, LmxGALK enzymatic activity was measured in the fractions corresponding to the homogenate and glycosomes, proving that it is active in promastigotes. In addition, it was shown that galactose cannot serve as an important carbon source for sustaining parasite growth, as cultures of promastigotes from three Leishmania species in LIT medium containing either no sugar or supplemented with D-galactose (20 mM) grew to lower density compared to these cultured with D-glucose (20 mM). These results suggest that D-galactose is mainly used for UDP-galactose synthesis by the salvage route, functioning when glucose is depleted from the medium, similar to the conditions promastigotes experience in the gut of the insect vector during its life cycle., Competing Interests: Declaration of competing interest Ángel E. Lobo-Rojas. “I have nothing to declare". María A. Delgado-Chacón. “I have nothing to declare". Edward A. Valera-Vera. “I have nothing to declare". Marirene Chacón-Arnaude. “I have nothing to declare". Mary Carmen Pérez-Aguilar. “I have nothing to declare". Rocío Rondón-Mercado. “I have nothing to declare". Ender Quintero-Troconis. “I have nothing to declare". Wilfredo Quiñones. “I have nothing to declare". Juan L. Concepción. “I have nothing to declare". Ana J. Cáceres. “I have nothing to declare"., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Protein phosphorylation is a fundamental cellular regulatory mechanism that governs the activation and deactivation of numerous proteins. In two-component signaling transduction pathways, the phosphorylation of response regulator proteins and their subsequent diffusion play pivotal roles in signal transmission. However, the impact of protein phosphorylation on their dispersion properties remains elusive. In this study, using the response regulator CheY in bacterial chemotaxis as a model, we performed comprehensive measurements of the spatial distributions and diffusion characteristics of CheY and phosphorylated CheY through single-molecule tracking within live cells. We discovered that phosphorylation significantly enhances diffusion and mitigates the constraining influence of the cell membrane on these proteins. Moreover, we observed that ATP-dependent fluctuations also promote protein diffusion and reduce the restraining effect of the cell membrane. These findings highlight important effects of phosphorylation beyond protein activation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

When stressed, cells synthesize di-adenosine polyphosphates (Ap n A), and cellular organisms also express proteins that degrade these compounds to release ATP. Most of these proteins are members of the nudix hydrolase superfamily, and several are involved in bacterial pathogenesis, neurodevelopment, and cancer. The goal of this project is to assist in the discovery of inhibitors of these enzymes that could be used to study Ap n A function and the cellular role of these nudix enzymes. Because these enzymes cleave Ap 4 A and Ap 5 A to produce ATP, two standard ATP detection techniques were optimized and compared here for their suitability for high throughput screening. In the first assay, cleavage is monitored by coupling to a reaction catalyzed by firefly luciferase. In the second assay, cleavage is detected by coupling to hexokinase, glucose 6-phosphate dehydrogenase, and diaphorase. Although the former assay was more sensitive, the latter was more reproducible, linear, and suitable for screening and kinetic analyses. The assays were used to characterize the kinetics of reactions catalyzed by various nudix enzymes isolated from E. coli, humans, and Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Results reveal subtle differences between the proteins that might be exploited to identify specific small molecule inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The HprSR constitutes the bacterial two-component regulatory system engaged by Escherichia coli to reduce the damaging effects of reactive chlorine and oxygen species present in its cytosol. Hypochlorous acid (HOCl) has been shown to be the molecule capable of activating of the HprSR system. HOCl is produced upon pathogen invasion by phagocytic cells of the human innate immune system, particularly neutrophils, to take advantage of its powerful antimicrobial attributes. Therefore, comprehensive studies concerning bacterial sensing and regulatory HprSR system are indispensable in understanding and effectively eliminating pathogens. Here we present the first crystal structure, solved at 1.7 Å resolution, of the HprS cytoplasmic domains arranged as a homodimer. In both protomers, the catalytic ATP-binding domain contains a non-hydrolysable ATP analog coordinated by a magnesium ion. This structure allowed us to provide a detailed characterization of kinase-substrate interaction. Furthermore, the structural data are supported by biophysical studies of kinase interaction with cognate response regulator HprR and substrate ATP. The kinase activity is also assessed in the presence or absence of HprR., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aim: The aim of the present study was to define whether cardiac myosin contributes to energy conservation in the heart of hibernating mammals., Methods: Thin cardiac strips were isolated from the left ventricles of active and hibernating grizzly bears; and subjected to loaded Mant-ATP chase assays, X-ray diffraction and proteomics., Main Findings: Hibernating grizzly bears displayed an unusually high proportion of ATP-conserving super-relaxed cardiac myosin molecules that are likely due to altered levels of phosphorylation and rod region stability., Conclusions: Cardiac myosin depresses the heart's energetic demand during hibernation by modulating its function., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julien Ochala reports financial support was provided by Carlsberg Foundation. Henk Granzier reports financial support was provided by National Institutes of Health. Weikang Ma reports financial support was provided by National Institutes of Health. Weikang Ma reports a relationship with Edgewise Therapeutics, Inc. that includes: consulting or advisory. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Hepatic ischemia-reperfusion injury (HIRI) is an important cause of liver injury following liver transplantation and major resections, and neutrophils are the key effector cells in HIRI. Double-negative T regulatory cells (DNT) are increasingly recognized as having critical regulatory functions in the immune system. Whether DNT expresses distinct immunoregulatory mechanisms to modulate neutrophils, as in HIRI, remains largely unknown. In this study, we found that murine and human DNT highly expressed CD39 that protected DNT from extracellular ATP-induced apoptosis and generated adenosine in tandem with CD73, to induce high levels of neutrophil apoptosis. Furthermore, extracellular adenosine enhanced DNT survival and suppressive function by upregulating survivin and NKG2D expression via the A2AR/pAKT/FOXO1 signaling pathway. Adoptive transfer of DNT ameliorated HIRI in mice through the inhibition of neutrophils in a CD39-dependent manner. Lastly, the adoptive transfer of A2ar -/- DNT validated the importance of adenosine/A2AR signaling, in promoting DNT survival and immunomodulatory function to protect against HIRI in vivo. In conclusion, purinergic signaling is crucial for DNT homeostasis in HIRI. Augmentation of CD39 or activation of A2AR signaling in DNT may provide novel therapeutic strategies to target innate immune disorders., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Macrophages play a crucial role in immune responses and undergo metabolic reprogramming to fulfill their functions. The tetramerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) induces the production of the anti-inflammatory cytokine interleukin (IL)-10 in vivo, but the underlying mechanism remains elusive. Here, we report that PKM2 activation with the pharmacological agent TEPP-46 increases IL-10 production in LPS-activated macrophages by metabolic reprogramming, leading to the production and release of ATP from glycolysis. The effect of TEPP-46 is abolished in PKM2-deficient macrophages. Extracellular ATP is converted into adenosine by ectonucleotidases that activate adenosine receptor A2a (A2aR) to enhance IL-10 production. Interestingly, IL-10 production induced by PKM2 activation is associated with improved mitochondrial health. Our results identify adenosine derived from glycolytic ATP as a driver of IL-10 production, highlighting the role of tetrameric PKM2 in regulating glycolysis to promote IL-10 production., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

During aerobic growth, S. aureus relies on acetate overflow metabolism, a process where glucose is incompletely oxidized to acetate, for its bioenergetic needs. Acetate is not immediately captured as a carbon source and is excreted as waste by cells. The underlying factors governing acetate overflow in S. aureus have not been identified. Here, we show that acetate overflow is favored due to a thermodynamic bottleneck in the TCA cycle specifically involving the oxidation of succinate to fumarate by succinate dehydrogenase. This bottleneck reduces flux through the TCA cycle, making it more efficient for S. aureus to generate ATP via acetate overflow metabolism. Additionally, the protein allocation cost of maintaining ATP flux through the restricted TCA cycle is greater than that of acetate overflow metabolism. Finally, we show that the TCA cycle bottleneck provides S. aureus the flexibility to redirect carbon toward maintaining redox balance through lactate overflow when oxygen becomes limiting, albeit at the expense of ATP production through acetate overflow. Overall, our findings suggest that overflow metabolism offers S. aureus distinct bioenergetic advantages over a thermodynamically constrained TCA cycle, potentially supporting its commensal-pathogenic lifestyle., Competing Interests: The authors declare no conflict of interest.

Background: Osteoporosis is a major public health concern characterized by decreased bone density. Among various therapeutic strategies, apoptotic extracellular vesicles (ApoEVs) have emerged as promising agents in tissue regeneration. Specifically, T cell-derived ApoEVs have shown substantial potential in facilitating bone regeneration. However, it remains unclear whether ApoEVs can promote bone mass recovery through enzymatic activity mediated by membrane surface molecules. Therefore, this study aimed to investigate whether T cell-derived ApoEVs could promote bone mass recovery in osteoporosis mice and reveal the underlying mechanisms., Methods: ApoEVs were isolated through sequential centrifugation, and their proteomic profiles were identified via mass spectrometry. Western blot and immunogold staining confirmed the enrichment of CD39 and CD73 on ApoEVs. The role of CD39 and CD73 in hydrolyzing adenosine triphosphate (ATP) to adenosine was evaluated by quantifying the levels of ATP and adenosine. Inhibitors of CD39 and CD73, and an A2BR antagonist were used to explore the molecular mechanism of ApoEVs in promoting bone regeneration., Results: ApoEVs significantly reduced bone loss and promote the osteogenic differentiation of BMMSCs in ovariectomy (OVX) mice. We observed increased levels of extracellular ATP and a decrease in CD39 and CD73, key enzymes in ATP-to-adenosine conversion in bone marrow of OVX mice. We found that ApoEVs are enriched with CD39 and CD73 on their membranes, which enable the hydrolysis of extracellular ATP to adenosine both in vitro and in vivo. The adenosine generated by ApoEVs inhibits the inflammatory response and promotes osteogenesis through A2BR and downstream PKA signaling., Conclusion: T cell-derived ApoEVs are enriched with CD39 and CD73, enabling them to hydrolyze extracellular ATP to adenosine, thereby promoting bone regeneration via A2BR and PKA signaling pathway. Our data underscore the substantive role of T cell-derived ApoEVs to treat osteoporosis, thus providing new ideas for the development of ApoEVs-based therapies in tissue regeneration., Competing Interests: The authors declare no competing interests in this work., (© 2025 Yang et al.)

The outer retina (OR) is highly energy demanding. Impaired energy metabolism combined with high demands are expected to cause energy insufficiencies that make the OR susceptible to complex blinding diseases such as age-related macular degeneration (AMD). Here, anatomical, physiological and quantitative molecular data were used to calculate the ATP expenditure of the main energy-consuming processes in three cell types of the OR for the night and two different periods during the day. The predicted energy demands in a rod dominated (perifovea) area are 1.69 x 1013 ATP/s/mm2 tissue in the night and 6.53 x 1012 ATP/s/mm2 tissue during the day with indoor light conditions. For a cone-dominated foveal area the predicted energy demands are 6.41 x 1012 ATP/s/mm2 tissue in the night and 6.75 x 1012 ATP/s/mm2 tissue with indoor light conditions during daytime. We propose the likely need for diurnal/circadian shifts in energy demands to efficiently stagger all energy consuming processes. Our data provide insights into vulnerabilities in the aging OR and suggest that diurnal constraints may be important when considering therapeutic interventions to optimize metabolism., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Kiel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Real-time monitoring of molecular transformations is crucial for advancements in biotechnology. In this study, we introduce a novel self-assembling 19 F-labeled nuclear magnetic resonance (NMR) probe that disassembles upon interaction with various nucleotides. This interaction not only activates the 19 F signals but also produces distinct signatures for each specific component, thereby enabling precise identification and quantification of molecules in evolving samples. We demonstrate the capability of this probe for real-time monitoring of adenosine triphosphate (ATP) hydrolysis and screening potential enzyme inhibitors. These applications highlight the probe's significant potential in enzyme analysis, drug development, and disease diagnostics., (© 2024 Wiley-VCH GmbH.)

Supramolecular assembly allows multiple chemical/bio-components integrated as one system for cascade biochemical reactions. Herein the graphitic carbon nitrides (g-C 3 N 4 ) as photocatalyst trapped in a dipeptide hydrogel covering adenosine triphosphate (ATP) synthase accelerates the photophosphorylation through ATP synthesis. Self-assembled N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) as nanofibrils to allow g-C 3 N 4 nanosheets are embedded as a complex Fmoc-FF/g-C 3 N 4 hydrogel. Fmoc-FF gel exhibits good electronic coupling with g-C 3 N 4 , which enables a photo-induced proton generation. The transmembrane proton gradient can be established by ATP synthase-lipid reconstituted on the surface of the Fmoc-FF/g-C 3 N 4 hydrogel to enhance the ATP synthesis. It indicates that the Fmoc-FF/g-C 3 N 4 /ATP synthase-lipid film can possess a longer-term ATP production capability and allow repeated immersion for sustained ATP production. Such a hydrogel-supported ATP synthesis platform is achieved by a procedure at a larger scale., (© 2024 Wiley-VCH GmbH.)

Recalcitrant bacterial infections can be caused by various types of dormant bacteria, including persisters and viable but nonculturable (VBNC) cells. Despite their clinical importance, we know fairly little about bacterial dormancy development and recovery. Previously, we established a correlation between protein aggregation and dormancy in Escherichia coli. Here, we present further support for a direct relationship between both. Our experiments demonstrate that aggregates progressively sequester proteins involved in energy production, thereby likely causing ATP depletion and dormancy. Furthermore, we demonstrate that structural features of protein aggregates determine the cell's ability to exit dormancy and resume growth. Proteins were shown to first assemble in liquid-like condensates that solidify over time. This liquid-to-solid phase transition impedes aggregate dissolution, thereby preventing growth resumption. Our data support a model in which aggregate structure, rather than cellular activity, marks the transition from the persister to the VBNC state., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

DEAD-box RNA-dependent ATPases are ubiquitous in all domains of life where they bind and remodel RNA and RNA-protein complexes. DEAD-box ATPases with helicase activity unwind RNA duplexes by local opening of helical regions without directional movement through the duplexes and some of these enzymes, including Ded1p from Saccharomyces cerevisiae, oligomerize to effectively unwind RNA duplexes. Whether and how DEAD-box helicases coordinate oligomerization and unwinding is not known and it is unclear how many base pairs are actively opened. Using high-resolution optical tweezers and fluorescence, we reveal a highly dynamic and stochastic process of multiple Ded1p protomers assembling on and unwinding an RNA duplex. One Ded1p protomer binds to a duplex-adjacent ssRNA tail and promotes binding and subsequent unwinding of the duplex by additional Ded1p protomers in 4-6 bp steps. The data also reveal rapid duplex unwinding and rezipping linked with binding and dissociation of individual protomers and coordinated with the ATP hydrolysis cycle., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions necessary for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin of replication, catalyzing the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. We report the cryo-electron microscopy structures of Rep68 bound to the adeno-associated virus integration site 1 in different nucleotide-bound states. In the nucleotide-free state, Rep68 forms a heptameric complex around DNA, with three origin-binding domains (OBDs) bound to the Rep-binding element sequence, while three remaining OBDs form transient dimers with them. The AAA+ domains form an open ring without interactions between subunits and DNA. We hypothesize that the heptameric structure is crucial for loading Rep68 onto double-stranded DNA. The ATPγS complex shows that only three subunits associate with the nucleotide, leading to a conformational change that promotes the formation of both intersubunit and DNA interactions. Moreover, three phenylalanine residues in the AAA+ domain induce a steric distortion in the DNA. Our study provides insights into how an SF3 helicase assembles on DNA and provides insights into the DNA melting process., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Helicase is a nucleic acid motor that catalyses the unwinding of double-stranded (ds) RNA and DNA via ATP hydrolysis. Helicases can act either as a nucleic acid motor that unwinds its ds substrates or as a chaperone that alters the stability of its substrates, but the two activities have not yet been reported to act simultaneously. Here, we used single-molecule techniques to unravel the synergistic coordination of helicase and chaperone activities, and found that the severe acute respiratory syndrome coronavirus helicase (nsp13) is capable of two modes of action: (i) binding of nsp13 in tandem with the fork junction of the substrate mechanically unwinds the substrate by an ATP-driven synchronous power stroke; and (ii) free nsp13, which is not bound to the substrate but complexed with ADP in solution, destabilizes the substrate through collisions between transient binding and unbinding events with unprecedented melting capability. Our findings provide new insights into how the same enzyme works via two modes on different parts of the substrate and synergistically catalyses the unwinding reaction, utilizing ATP and recycling its by-product ADP as an energy source., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Long-term durable remission in patients with B cell malignancies following chimeric antigen receptor (CAR)-T cell immunotherapy remains unsatisfactory, often due to antigen escape. Malignant B cell transformation and oncogenic growth relies on efficient ATP synthesis, although the underlying mechanisms remain unclear. Here, we report that YTHDF2 facilitates energy supply and antigen escape in B cell malignancies, and its overexpression alone is sufficient to cause B cell transformation and tumorigenesis. Mechanistically, YTHDF2 functions as a dual reader where it stabilizes mRNAs as a 5-methylcytosine (m 5 C) reader via recruiting PABPC1, thereby enhancing their expression and ATP synthesis. Concomitantly, YTHDF2 also promotes immune evasion by destabilizing other mRNAs as an N 6 -methyladenosine (m 6 A) reader. Small-molecule-mediated targeting of YTHDF2 suppresses aggressive B cell malignancies and sensitizes them to CAR-T cell therapy., Competing Interests: Declaration of interests Z.C., X.D., and J.C. have filed a patent application related to this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sublethal tumor cells have an urgent need for energy, making it common for them to switch metabolic phenotypes between glycolysis and oxidative phosphorylation (OXPHOS) for compensatory energy supply; thus, the synchronous interference of dual metabolic pathways for limiting energy level is essential in inhibiting sublethal tumor growth. Herein, a multifunctional nanoplatform of Co-MOF-loaded anethole trithione (ADT) and myristyl alcohol (MA), modified with GOx and hyaluronic acid (HA) was developed, namely, CAMGH. It could synchronously interfere with dual metabolic pathways including glycolysis and OXPHOS to restrict the adenosine triphosphate (ATP) supply, achieving the inhibition to sublethal tumors after microwave (MW) thermal therapy. Under low-power MW irradiation, CAMGH induced certain tumor thermal damage while ensuring the safety of the surrounding normal tissues. The loaded GOx consumed glucose in tumors, undoubtedly blocking the main energy supply pathway, the glycolytic pathway. Then, H 2 O 2 generated from GOx reacted with Co 2+ to produce cytotoxic ·OH, combining with the released H 2 S from ADT to co-obstruct OXPHOS and then synergy with the above glycolysis blocking for a more effective ATP inhibition. The powerful depletion of ATP caused significant suppression of damage resistance protein upregulated after thermal stimulation, i.e., HSP90, and then the activation of caspase-3, achieving the simultaneous reversal of heat resistance and apoptosis resistance. Altogether, the CAMGH-based synchronous interference of dual metabolic pathways shows the potential to break down tumor self-repair, presenting an alternative strategy to enhance the therapeutic effect of tumor MW thermal therapy.

Background: The cryoinjury of semen during cryopreservation reduces sperm motility, constraining the application of artificial insemination (AI) in bovine reproduction. Some fertility markers, related to sperm motility before and after freezing have been identified. However, little is known about the biological mechanism through which freezing reduces sperm motility. This study investigated the selective effects of cryoinjury on high-motility sperm (HMS) and low-motility sperm (LMS) in frozen-thawed from the perspectives of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP levels. The molecular mechanism of decreased sperm motility caused by cryoinjury was explored through a joint analysis of 4D-label free quantitative proteomics and non-targeted metabolomics., Results: The results indicate that low levels of ROS and high degrees of MMP and ATP play a critical role in the survival of HMS during the freezing process. The sperm samples from the frozen-thawed HMS and LMS were analysed for proteomics and metabolomics, 2,465 proteins and 4,135 metabolites were detected in bovine sperm samples. In contrast to LMS, HMS have 106 proteins and 106 metabolites with high abundance expression, and 79 proteins and 223 metabolites with low abundance expression. Proteomics and metabolomics data exhibit that highly expressed antioxidant enzymes and metabolites in HMS can maintain sperm motility by regulating the ROS produced during freezing to prevent sperm from oxidative stress and apoptosis. Furthermore, the KEGG analysis of differential proteins and metabolites during the freezing process implies that the significant enrichment of glycolysis and cAMP in HMS can guarantee energy supply., Conclusions: The results provided that during the process of bovine sperm freezing, highly expressed antioxidant enzymes can regulate the reactive oxygen species levels to avoid oxidative stress and the glycolysis signalling pathway ensures ATP production can sustain frozen-thawed sperm motility., Competing Interests: Declarations. Ethics approval and consent to participate: All animals were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China, and the study was approved by The Animal Administration and Ethics Committee of Qingdao Agricultural University, Animal Science and Technology College (Permit No. 20230610). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Microglia and the border-associated macrophages contribute to the modulation of cerebral blood flow, but the mechanisms have remained uncertain. Here, we show that microglia regulate the cerebral blood flow baseline and the responses to whisker stimulation or intra-cisternal magna injection of adenosine triphosphate, but not intra-cisternal magna injection of adenosine in mice model. Notably, microglia repopulation corrects these cerebral blood flow anomalies. The microglial-dependent regulation of cerebral blood flow requires the adenosine triphosphate-sensing P2RY12 receptor and ectonucleotidase CD39 that initiates the dephosphorylation of extracellular adenosine triphosphate into adenosine in both male and female mice. Pharmacological inhibition or CX3CR1-CreER-mediated deletion of CD39 mimics the cerebral blood flow anomalies in microglia-deficient mice and reduces the upsurges of extracellular adenosine following whisker stimulation. Together, these results suggest that the microglial CD39-initiated breakdown of extracellular adenosine triphosphate co-transmitter is an important step in neurovascular coupling and the regulation of cerebrovascular reactivity., Competing Interests: Competing interests: S.C.R. is a scientific founder of Purinomia Biotech Inc and has consulted for eGenesis and SynLogic Inc; his interests are reviewed and managed by HMFP, Beth Israel Deaconess Medical Center by the institutional conflict-of-interest policies. The other authors declare no competing interests., (© 2025. The Author(s).)

Xanthomonas citri pv. malvacearum (Xcm) associated with bacterial blight disease is a significant and widespread pathogen affecting cotton worldwide. The excessive use of harmful chemicals to control plant pathogens has exerted a negative impact on environmental safety. Thymol is a monoterpene phenol present in the essential oils of plants belonging to Lamiaceae family. In this study the antimicrobial activity of thymol was evaluated against Xcm. The minimum inhibitory concentration (MIC) and 99.9% bactericidal concentration (MBC) of thymol against Xcm were 2 and 4 mg/mL, respectively. The effect of MIC and MBC of thymol against Xcm was assessed on the Luria-Bertani medium. The effect of thymol on intercellular ATP levels, membrane potential, and motility in Xcm was assessed using fluorescence spectrometry for membrane potential and firefly luciferase-based assay for ATP levels. Thymol ruptured the cellular membrane of Xcm, resulting in decreased intracellular ATP concentrations, intracellular leakage of genetic material, and changes in membrane potential. Scanning electron microscopy images supported the impact of thymol on the cell membrane of Xcm. Moreover, thymol inhibited the swimming motility and biofilm formation of Xcm at concentrations equal to or above the MIC and MBC. In contrast, sub-MIC concentrations of thymol had little to no impact on the virulence of Xcm. In conclusion, thymol demonstrated the potential as a strong bactericidal compound against Xcm., Competing Interests: Declarations. Conflict of interest: The authors disclose no competing interest. Ethical Approval: Our research did not include any human subjects and animal experiments. Consent to Participate: Not applicable. Consent for Publication: Not applicable., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitochondrial uncouplers dissipate proton gradients and deplete ATP production from oxidative phosphorylation (OXPHOS). While the growth of prostate cancer depends on OXPHOS-generated ATP, the oncogenic pathway mediated by the transcription factor E2F1 is crucial for the progression of this deadly disease. Here, we report that mitochondrial uncouplers, including tizoxanide (TIZ), the active metabolite of the Food and Drug Administration (FDA)-approved anthelmintic nitazoxanide (NTZ), inhibit E2F1-mediated expression of genes involved in cell cycle progression, DNA synthesis, and lipid synthesis. Consequently, NTZ/TIZ induces S-phase kinase-associated protein 2 (SKP2)-mediated G1 arrest while impeding DNA synthesis, lipogenesis, and the growth of prostate cancer cells. The anti-cancer activity of TIZ correlates with its OXPHOS-uncoupling activity. NTZ/TIZ appears to inhibit ATP production, thereby activating the AMP-activated kinase (AMPK)-p38 pathway, leading to cyclin D1 degradation, Rb dephosphorylation, and subsequent E2F1 inhibition. Our results thus connect OXPHOS uncoupling to the inhibition of an essential oncogenic pathway, supporting repositioning NTZ and other mitochondrial uncouplers for prostate cancer therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

P2X receptors (P2XRs) are adenosine 5'-triphosphate (ATP)-gated ion channels comprising homomeric and heteromeric trimers of seven subtypes (P2X1-P2X7) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-electron microscopy structures of full-length wild-type human P2X4 receptor in apo closed, antagonist-bound inhibited, and ATP-bound desensitized states. Because the apo closed and antagonist-bound inhibited state structures of this slow-desensitizing P2XR include an intact cytoplasmic cap while the ATP-bound desensitized state structure does not, the cytoplasmic cap is formed before agonist binding. Furthermore, structural and functional data suggest the cytoplasmic cap is stabilized by lipids to modulate desensitization, and P2X4 is modified by glycosylation and palmitoylation. Last, our antagonist-bound inhibited state structure reveals features specific to the allosteric ligand-binding pocket in human receptors that facilitates development of small-molecule modulators.

Neurite degeneration (ND) precedes cell death in many neurodegenerative diseases. However, it remains unclear how this compartmentalized cell death process is orchestrated in the central nervous system (CNS). The establishment of a CNS axotomy model (using modified 3D LUHMES cultures) allowed us to study metabolic control of ND in human midbrain-derived neurons without the use of toxicants or other direct disturbance of cellular metabolism. Axotomy lead to a loss of the NAD + synthesis enzyme NMNAT2 within 2 h and a depletion of NAD + within 4-6 h. This process appeared specific, as isolated neurites maintained ATP levels and a coupled mitochondrial respiration for at least 6 h. In the peripheral nervous system (PNS) many studies observed that NAD + metabolism, in particular by the NADase SARM1, plays a major role in the ND occurring after axotomy. Since neither ferroptosis nor necroptosis, nor caspase-dependent apoptosis seemed to be involved in neurite loss, we investigated SARM1 as potential executioner (or controller). Knock-down or expression of a dominant-negative isoform of SARM1 indeed drastically delayed ND. Various modifications of NAD + metabolism known to modulate SARM1 activity showed the corresponding effects on ND. Moreover, supplementation with NAD + attenuated ND. As a third approach to investigate the role of altered NAD + metabolism, we made use of the WLD(s) protein, which has been found in a mutant mouse to inhibit Wallerian degeneration of axons. This protein, which has a stable NMNAT activity, and thus can buffer the loss of NMNAT2, protected the neurites by stabilizing neurite NAD + levels. Thus CNS-type ND was tightly linked to neurite metabolism in multiple experimental setups. Based on this knowledge, several new strategies for treating neurodegenerative diseases can be envisaged., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval: The work with the human cell line LUHMES has been approved by the University of Konstanz institutional review board. This statement confirmed that all methods were performed in accordance with the relevant guidelines and regulations (Statement number IRB21KN040-05/w). No other cell sources were used in this study and neither animal data nor data from human subjects were obtained in this study., (© 2025. The Author(s).)

The MCM motor of the eukaryotic replicative helicase is loaded as a double hexamer onto DNA by the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ATP binding supports formation of the ORC-Cdc6-Cdt1-MCM (OCCM) helicase-recruitment complex where ORC-Cdc6 and one MCM hexamer form two juxtaposed rings around duplex DNA. ATP hydrolysis by MCM completes MCM loading but the mechanism is unknown. Here, we used cryo-EM to characterise helicase loading with ATPase-dead Arginine Finger variants of the six MCM subunits. We report the structure of two MCM complexes with different DNA grips, stalled as they mature to loaded MCM. The Mcm2 Arginine Finger-variant stabilises DNA binding by Mcm2 away from ORC/Cdc6. The Arginine Finger-variant of the neighbouring Mcm5 subunit stabilises DNA engagement by Mcm5 downstream of the Mcm2 binding site. Cdc6 and Orc1 progressively disengage from ORC as MCM translocates along DNA. We observe that duplex DNA translocation by MCM involves a set of leading-strand contacts by the pre-sensor 1 ATPase hairpins and lagging-strand contacts by the helix-2-insert hairpins. Mutating any of the MCM residues involved impairs high-salt resistant DNA binding in vitro and double-hexamer formation assessed by electron microscopy. Thus, ATPase-powered duplex DNA translocation away from ORC underlies MCM loading., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The "Protein synthesis Using Recombinant Elements" ("PURE") system is a minimal biochemical system capable of carrying out cell-free protein synthesis using defined enzymatic components. This study extends PURE by integrating an ATP regeneration system based on pyruvate oxidase, acetate kinase, and catalase. The new pathway generates acetyl phosphate from pyruvate, phosphate, and oxygen, which is used to rephosphorylate ATP in situ . Successful ATP regeneration requires a high initial concentration of ∼10 mM phosphate buffer, which surprisingly does not affect the protein synthesis activity of PURE. The pathway can function independently or in combination with the existing creatine-based system in PURE; the combined system produces up to 233 μg/mL of mCherry, an enhancement of 78% compared to using the creatine system alone. The results are reproducible across multiple batches of homemade PURE and importantly also generalize to commercial systems such as PURExpress from New England Biolabs. These results demonstrate a rational bottom-up approach to engineering PURE, paving the way for applications in cell-free synthetic biology and synthetic cell construction.

Bioluminescence imaging (BLI) is a powerful, noninvasive imaging method for animal studies. NanoLuc luciferase and its derivatives are attractive bioluminescent reporters recognized for their efficient photon production and ATP independence. However, utilizing them for animal imaging poses notable challenges. Low substrate solubility has been a prominent problem, limiting in vivo brightness, while the susceptibility of luciferins to auto-oxidation by molecular oxygen in air increases handling complexity and poses an obstacle to obtaining consistent results. To address these issues, we developed a range of caged PEGylated luciferins with increased auto-oxidation resistance and water solubility of up to 25 mM, resulting in substantial in vivo bioluminescence increases in mouse models. This advancement has created the brightest and most sensitive luciferase-luciferin combination, enabling high-speed video-rate imaging of freely moving mice with brain-expressed luciferase. These innovative substrates offer new possibilities for investigating a wide range of biological processes and are poised to become invaluable resources for chemical, biological, and biomedical fields.

Objectives: Mitochondrial Ca 2+ uniporter (MCU) provides a Ca 2+ influx pathway from the cytosol into the mitochondrial matrix and a moderate mitochondrial Ca 2+ rise stimulates ATP production and cell growth. MCU is highly expressed in various cancer cells including breast cancer cells, thereby increasing the capacity of mitochondrial Ca 2+ uptake, ATP production, and cancer cell proliferation. The objective of this study was to examine MCU inhibition as an anti-cancer mechanism., Methods: The effects of MCU-i4, a newly developed MCU inhibitor, on cell viability, apoptosis, cytosolic Ca 2+ , mitochondrial Ca 2+ and potential, glycolytic rate, generation of ATP, and reactive oxygen species, were examined in breast cancer BT474 cells., Results: MCU-i4 caused apoptotic cell death, and it decreased and increased, respectively, mitochondrial and cytosolic Ca 2+ concentration. Inhibition of MCU by MCU-i4 revealed that cytosolic Ca 2+ elevation resulted from endoplasmic reticulum (ER) Ca 2+ release via inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RYR). Unexpectedly, MCU-i4 enhanced glycolysis and ATP production; it also triggered a large production of reactive oxygen species (ROS) and mitochondrial membrane potential collapse., Conclusion: Cytotoxic mechanisms of MCU-i4 in cancer cells involved enhanced glycolysis and heightened formation of ATP and ROS. It is conventionally believed that cancer cell death could be caused by inhibition of glycolysis. Our observations suggest cancer cell death could also be induced by increased glycolytic metabolism., Competing Interests: The authors declare no conflicts of interest to report regarding the present study., (© 2025 The Authors.)

The pectic polysaccharide WTRP-A0.2b (43 kDa) has been isolated from Typhonii rhizoma and analyzed in terms of its structural features, anti-tumor activities and mechanism of action. NMR, FT-IR, monosaccharide composition, and enzymology demonstrate that WTRP-A0.2b is composed of rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II) and homogalacturonan (HG) domains with mass ratios of 3.7:1:1.7, respectively. The RG-I domains contain a highly branched structure that is substituted primarily with β-D-1,4-galactan, α-L-1,5-arabinan, and AG-II. The HG domains contain un-esterified and methyl-esterified and/or acetyl-esterified oligogalacturonides with a degree of polymerization of 1-8. In vitro experiments demonstrate that WTRP-A0.2b inhibits proliferation of K562 cells by inducing mitochondrial damage and suppressing glycolysis. This activity promotes mitochondrial permeability, increases production of reactive oxygen species (ROS), boosts extracellular oxygen consumption and adenosine triphosphate (ATP) content, while it decreases uncoupling protein-2 (UCP2) expression and lactic acid content. Our results provide valuable insight for screening natural polysaccharide-based anti-tumor effects of polysaccharides from Typhonii rhizoma., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)