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P2X receptors are trimeric ion channels activated by adenosine triphosphate (ATP) that contribute to pathophysiological processes ranging from asthma to neuropathic pain and neurodegeneration. A number of small-molecule antagonists have been identified for these important pharmaceutical targets. However, the molecular pharmacology of P2X receptors is poorly understood because of the chemically disparate nature of antagonists and their differential actions on the seven constituent subtypes. Here, we report high-resolution cryo-electron microscopy structures of the homomeric rat P2X 7 receptor bound to five previously known small-molecule allosteric antagonists and a sixth antagonist that we identify. Our structural, biophysical, and electrophysiological data define the molecular determinants of allosteric antagonism in this pharmacologically relevant receptor, revealing three distinct classes of antagonists that we call shallow, deep, and starfish. Starfish binders, exemplified by the previously unidentified antagonist methyl blue, represent a unique class of inhibitors with distinct functional properties that could be exploited to develop potent P2X 7 ligands with substantial clinical impact.

Rapid elimination of paternal mitochondria following fertilization is a conserved event in most animals, but its physiological significance remains unclear. We find that modest delay of paternal mitochondrial elimination (PME) in Caenorhabditis elegans embryos unexpectedly impairs mating and cognition of adult animals and alters their locomotion behaviors. Delayed PME causes decreased adenosine triphosphate (ATP) levels in early embryos, which lead to impaired physiological functions of adult animals through an energy-sensing pathway mediated by an adenosine monophosphate (AMP)-activated protein kinase, AAK-2, and a forkhead box class O (FOXO) transcription factor, DAF-16. Treatment of PME-delayed animals with MK-4, a subtype of vitamin K 2 that can improve mitochondrial ATP production, restores ATP levels in early embryos, and rescues physiological defects of adult animals. Our results suggest that moderate PME delay during embryo development adversely affects crucial physiological functions in adults, which could be evolutionarily disadvantageous. These observations provide mechanistic explanations for the need to swiftly remove paternal mitochondria early during embryo development.

Antibiotic resistance is a major global challenge requiring new treatments and a better understanding of the bacterial resistance mechanisms. In this study, we compared ampicillin-resistant (R-AMP) and gentamicin-resistant (R-GEN) Staphylococcus aureus strains with a sensitive strain (ATCC6538) using metabolomics. We identified 109 metabolites; 28 or 31 metabolites in R-AMP or R-GEN differed from those in ATCC6538. Moreover, R-AMP and R-GEN were enriched in five and four pathways, respectively. R-AMP showed significantly up-regulated amino acid metabolism and down-regulated energy metabolism, whereas R-GEN exhibited an overall decrease in metabolism, including carbohydrate, energy, and amino acid metabolism. Furthermore, the activities of the metabolism-related enzymes pyruvate dehydrogenase and TCA cycle dehydrogenases were inhibited in antibiotic-resistant bacteria. Significant decreases in NADH and ATP levels were also observed. In addition, the arginine biosynthesis pathway, which is related to nitric oxide (NO) production, was enriched in both antibiotic-resistant strains. Enhanced NO synthase activity in S. aureus promoted NO production, which further reduced reactive oxygen species, mediating the development of bacterial resistance to ampicillin and gentamicin. This study reveals that bacterial resistance affects metabolic profile, and changes in energy metabolism and arginine biosynthesis are important factors leading to drug resistance in S. aureus .

F-type ATP synthases are extensively researched protein complexes because of their widespread and central role in energy metabolism. Progress in structural biology, proteomics, and molecular biology has also greatly advanced our understanding of the catalytic mechanism, post-translational modifications, and biogenesis of chloroplast ATP synthases. Given their critical role in light-driven ATP generation, tailoring the activity of chloroplast ATP synthases and modeling approaches can be applied to modulate photosynthesis. In the future, advances in genetic manipulation and protein design tools will significantly expand the scope for testing new strategies in engineering light-driven nanomotors., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Tumor-associated antigens (TAAs) are not exclusively expressed in cancer cells, inevitably causing the "on target, off tumor" effect of molecular recognition tools. To achieve precise recognition of cancer cells, by using protein tyrosine kinase 7 (PTK7) as a model TAA, a DNA molecular logic circuit Aisgc8 was rationally developed by arranging H + -binding i-motif, ATP-binding aptamer, and PTK7-targeting aptamer Sgc8c in a DNA sequence. Aisgc8 output the conformation of Sgc8c to recognize PTK7 on cells in a simulated tumor microenvironment characterized by weak acidity and abundant ATP, but not in a simulated physiological environment. Through in vitro and in vivo results, Aisgc8 demonstrated its ability to precisely recognize cancer cells and, as a result, displayed excellent performance in tumor imaging. Thus, our studies produced a simple and efficient strategy to construct DNA logic circuits, opening new possibilities to develop convenient and intelligent precision diagnostics by using DNA logic circuits.

Temporal control over self-assembly processes is a highly desirable attribute that is efficiently exhibited by biological systems, such as actin filaments. In nature, various proteins undergo enzymatically catalysed chemical reactions that kinetically govern their structural and functional properties. Consequently, any stimuli that can alter their reaction kinetics can lead to a change in their growth or decay profiles. This underscores the urgent need to investigate bioinspired, adaptable and controllable synthetic materials. Herein we intend to develop a general strategy for controlling the growth and decay of self-assembled systems via enzymatically coupled reactions. We achieve this by the coupling of enzymes phosphokinase/phosphatase with a bolaamphiphilic cationic chromophore (PDI) which selectively self-assembles with ATP and disassembles upon its enzymatic hydrolysis. The aggregation process is efficiently regulated by the controlled in situ generation of ATP, through enzymatic reactions. By carefully managing the ATP generating components, we realize precise control over the self-assembly process. Moreover, we also show self-assembled structures with programmed temporal decay profiles through coupled enzymatic reactions of ATP generation and hydrolysis, essentially rendering the process dissipative. This work introduces a novel strategy to generate a reaction-coupled one-dimensional nanostructure with controlled dimensions inspired by biological systems.

Priority 1: critical WHO pathogen Acinetobacter baumannii depends on ATP synthesis and ATP:ADP homeostasis and its bifunctional F 1 F O -ATP synthase. While synthesizing ATP, it regulates ATP cleavage by its inhibitory ε subunit to prevent wasteful ATP consumption. We determined cryo-electron microscopy structures of the ATPase active A. baumannii F 1 -αßγε Δ134-139 mutant in four distinct conformational states, revealing four transition states and structural transformation of the ε's C-terminal domain, forming the switch of an ATP hydrolysis off- and an ATP synthesis on-state based. These alterations go in concert with altered motions and interactions in the catalytic- and rotary subunits of this engine. These A. baumannii interacting sites provide novel pathogen-specific targets for inhibitors, with the aim of ATP depletion and/or ATP synthesis and growth inhibition. Furthermore, the presented diversity to other bacterial F-ATP synthases extends the view of structural elements regulating such a catalyst., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Ehrlichiosis is a potentially life-threatening disease caused by infection with the obligatory intracellular bacteria Ehrlichia species. Ehrlichia japonica infection of mice provides an animal model of ehrlichiosis as it recapitulates full-spectrum and lethal ehrlichiosis in humans. The E. japonica transposon mutant of EHF0962 , which encodes a previously uncharacterized hypothetical protein, is attenuated in both infection and virulence in mice. EHF0962 was hence named here as resistance-inducing protein of Ehrlichia (RipE). Using this Δ ripE mutant, we studied how RipE protein contributes to Ehrlichia pathogenesis. Ehrlichia species have an intracellular developmental cycle and a brief extracellular stage to initiate a new cycle of infection. Majority of RipE proteins were expressed on the surface of the smaller infectious dense-core stage of bacteria. Extracellular Δ ripE E. japonica contained significantly less adenosine triphosphate (ATP) and lost infectivity more rapidly in culture compared with wild-type (WT) E. japonica . Genetic complementation in the Δ ripE mutant or overexpression of ripE in WT E. japonica significantly increased bacterial ATP levels, and RipE-overexpressing E. japonica was more virulent in mice than WT E. japonica . RipE is conserved among Ehrlichia species. Immunization of mice with recombinant RipE induced an in vitro infection-neutralizing antibody, significantly prolonged survival time after a lethal dose of E. japonica challenge, and cross-protected mice from infection by Ehrlichia chaffeensis , the agent of human monocytic ehrlichiosis. Our findings shed light on the extracellular stage of Ehrlichia , highlighting the importance of RipE and ATP levels in Ehrlichia for extracellular resistance and the next cycle of infection. Thus, RipE is a critical Ehrlichia protein for infection as such can be a potential vaccine target for ehrlichiosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Chien, Lin, Duan, Denton, Kawahara and Rikihisa.)

Using Phosphorus Magnetic Resonance Spectroscopy ( 31 P MRS), we examined five metabolites associated with brain energy cycle, and cellular membrane production and degradation in 11 brain regions of 48 children (age 6-15), and 80 middle-aged and older adults (age 52-87). Levels of phosphomonoesters (PMEs) and phosphodiesters (PDEs), gamma plus alpha adenosine triphosphate (γαATP), phosphocreatine (PCr) and inorganic phosphate (Pi), were residualized on the total amplitude value. PMEs were greater in children compared to adults, whereas PDEs showed the opposite age difference. Higher γαATP and lower Pi were found in children compared to adults. The age group differences were particularly salient in the association cortices and anterior white matter. Among children, age correlated negatively with PMEs and positively with PDEs in association cortices. Compared to children, adults had lower intracellular pH. The results suggest reduction in membrane synthesis and increase in membrane degradation in adolescents and to a greater degree in adults compared to younger children. Concomitant reduction in γαATP and increase in Pi are consistent with reduced energy consumption in adolescents and further drop in middle-aged and older adults, although it is impossible to distinguish declines in energy supply from reduced demand due to shrinking neuropil, without longitudinal studies., (© 2024 The Author(s). Human Brain Mapping published by Wiley Periodicals LLC.)

Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins 1-3 . The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles 4,5 . Here we report that the plant ESCRT component FREE1 6 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization., (© 2024. The Author(s).)

P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design., (© 2024. The Author(s).)

Background: Purinergic receptor P2X4 (P2X4R), highly expressed on microglia and macrophages, is activated by ATP released from damaged cells and linked to poststroke inflammation. Previous studies showed that short-term P2X4R inhibition reduces inflammation and promotes long term recovery, but the mechanism underlying P2X4R and inflammation remains unclear. We hypothesized that P2X4R absence or pharmacological blockade can enhance macrophage phagocytic function by alleviating excessive inflammation after stroke., Methods and Results: We divided P2X4R knockout and littermate control mice into 2 groups either naive or mice subjected to ischemic stroke surgery. Additionally, the regular WT mice subjected to ischemic stroke were treated with 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one BD (a P2X4R inhibitor) or vehicle. We isolated phagocytic cells from mice in each group and assayed phagocytic activity by quantifying uptake of fluorescent beads and bioparticles using flow cytometry or confocal microscopy and by measuring protein expression related to phagocytosis. Short-term inhibition of P2X4R with with 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one treatment upregulated ANXA1 (annexinA1). P2X4R absence prevented ATP-induced decline in phagocytic uptake in macrophages. Microglia or macrophages derived from P2X4R knockout mice showed significantly increased phagocytic activity compared with microglia/macrophages taken from littermate control mice. Cell surface expression of CD36, a scavenger receptor protein, increased after stroke, and was higher in P2X4R knockout mice., Conclusions: This study suggests that blockade or absence of P2X4R increases phagocytic uptake of damaged tissue following ischemic stroke. Taken together with previous reports detailing how P2X4R inhibition is protective following stroke, our results demonstrate P2X4R may mediate long-term resolution after ischemic stroke by enhancing phagocytic clearance.

L-lactate modifies proteins through lactylation 1 , but how this process occurs is unclear. Here we identify the alanyl-tRNA synthetases AARS1 and AARS2 (AARS1/2) as intracellular L-lactate sensors required for L-lactate to stimulate the lysine lactylome in cells. AARS1/2 and the evolutionarily conserved Escherichia coli orthologue AlaRS bind to L-lactate with micromolar affinity and they directly catalyse L-lactate for ATP-dependent lactylation on the lysine acceptor end. In response to L-lactate, AARS2 associates with cyclic GMP-AMP synthase (cGAS) and mediates its lactylation and inactivation in cells and in mice. By establishing a genetic code expansion orthogonal system for lactyl-lysine incorporation, we demonstrate that the presence of a lactyl moiety at a specific cGAS amino-terminal site abolishes cGAS liquid-like phase separation and DNA sensing in vitro and in vivo. A lactyl mimetic knock-in inhibits cGAS, whereas a lactyl-resistant knock-in protects mice against innate immune evasion induced through high levels of L-lactate. MCT1 blockade inhibits cGAS lactylation in stressed mice and restores innate immune surveillance, which in turn antagonizes viral replication. Thus, AARS1/2 are conserved intracellular L-lactate sensors and have an essential role as lactyltransferases. Moreover, a chemical reaction process of lactylation targets and inactivates cGAS., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The epsilon toxin (Etx) from Clostridium perfringens has been identified as a potential trigger of multiple sclerosis, functioning as a pore-forming toxin that selectively targets cells expressing the plasma membrane (PM) myelin and lymphocyte protein (MAL). Previously, we observed that Etx induces the release of intracellular ATP in sensitive cell lines. Here, we aimed to re-examine the mechanism of action of the toxin and investigate the connection between pore formation and ATP release. We examined the impact of Etx on Xenopus laevis oocytes expressing human MAL. Extracellular ATP was assessed using the luciferin-luciferase reaction. Activation of calcium-activated chloride channels (CaCCs) and a decrease in the PM surface were recorded using the two-electrode voltage-clamp technique. To evaluate intracellular Ca 2+ levels and scramblase activity, fluorescent dyes were employed. Extracellular vesicles were imaged using light and electron microscopy, while toxin oligomers were identified through western blots. Etx triggered intracellular Ca 2+ mobilization in the Xenopus oocytes expressing hMAL, leading to the activation of CaCCs, ATP release, and a reduction in PM capacitance. The toxin induced the activation of scramblase and, thus, translocated phospholipids from the inner to the outer leaflet of the PM, exposing phosphatidylserine outside in Xenopus oocytes and in an Etx-sensitive cell line. Moreover, Etx caused the formation of extracellular vesicles, not derived from apoptotic bodies, through PM fission. These vesicles carried toxin heptamers and doughnut-like structures in the nanometer size range. In conclusion, ATP release was not directly attributed to the formation of pores in the PM, but to scramblase activity and the formation of extracellular vesicles., (© 2024 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Miltefosine is the first and only drug approved for the treatment of leishmaniasis. It is also known as a PI3K/AKT signaling pathway inhibitor utilized in anti-cancer or anti-viral therapies. However, the impact of miltefosine on male fertility has not been fully understood. Therefore, this study was performed to investigate the effects of miltefosine on sperm function during capacitation. Duroc spermatozoa were exposed to 0, 2.5, 5, 10, 20, 40, and 80 μM miltefosine and induced for capacitation. Our results showed that miltefosine dramatically increased the expression of PI3K/AKT signaling pathway-associated proteins. Sperm motility, motion kinetics, capacitation, and tyrosine phosphorylation were significantly suppressed by miltefosine. However, intracellular ATP levels and cell viability were not significantly affected. Our findings suggest that miltefosine may disrupt sperm function by abnormally increasing the levels of PI3K/AKT signaling pathway-associated proteins. Therefore, the harmful effects of miltefosine on male reproduction should be considered when using this drug., 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 B.V. All rights reserved.)

Neurons of the subpostremal nucleus of the solitary tract (NTS) respond to changes in extracellular glucose with alterations in membrane potential with both depolarization and hyperpolarization. From 5 mM glucose, a rapid shift to 0.5 mM glucose produces a membrane depolarization by an unknown mechanism in most neurons. However, the mechanism involved in this response needs to be known. Here, we investigated if the low glucose-induced depolarization could be mimicked by reducing ATP synthesis and possible mediators of this effect. We showed that applying the mitochondrial uncoupler CCCP (1 µM) reproduced the effects of low glucose depolarizing the membrane, generating an inward current, and decreasing membrane resistance. On the other hand, activation of AMPK did not alter these parameters. To test if low glucose and CCCP could depolarize the membrane by affecting the ionic gradient, we inhibited the electrogenic Na/K pump with 10 µM of ouabain. We observed a similar membrane depolarization but not a decrease in membrane resistance. We conclude that perfusion of neurons of the subpostremal NTS with a low glucose solution depolarizes the membrane by probably reducing intracellular ATP, but not by activating AMPK or decreasing the ionic gradient across the membrane., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Nonvesicular lipid trafficking pathways are an important process in every domain of life. The mechanisms of these processes are poorly understood in part due to the difficulty in kinetic characterization. One important class of glycolipids, lipopolysaccharides (LPS), are the primary lipidic component of the outer membrane of Gram-negative bacteria. LPS are synthesized in the inner membrane and then trafficked to the cell surface by the l ipo p olysaccharide t ransport proteins, Lpt B 2 FGCADE. By characterizing the interaction of a fluorescent probe and LPS, we establish a quantitative assay to monitor the flux of LPS between proteoliposomes on the time scale of seconds. We then incorporate photocaged ATP into this system, which allows for light-based control of the initiation of LPS transport. This control allows us to measure the initial rate of LPS transport (3.0 min -1 per LptDE). We also find that the rate of LPS transport by the Lpt complex is independent of the structure of LPS. In contrast, we find the rate of LPS transport is dependent on the proper function of the LptDE complex. Mutants of the outer membrane Lpt components, LptDE, that cause defective LPS assembly in live cells display attenuated transport rates and slower ATP hydrolysis compared to wild type proteins. Analysis of these mutants reveals that the rates of ATP hydrolysis and LPS transport are correlated such that 1.2 ± 0.2 ATP are hydrolyzed for each LPS transported. This correlation suggests a model where the outer membrane components ensure the coupling of ATP hydrolysis and LPS transport by stabilizing a transport-active state of the Lpt bridge.

Brain function requires a constant supply of glucose. However, the brain has no known energy stores, except for glycogen granules in astrocytes. In the present study, we report that continuous oligodendroglial lipid metabolism provides an energy reserve in white matter tracts. In the isolated optic nerve from young adult mice of both sexes, oligodendrocytes survive glucose deprivation better than astrocytes. Under low glucose, both axonal ATP levels and action potentials become dependent on fatty acid β-oxidation. Importantly, ongoing oligodendroglial lipid degradation feeds rapidly into white matter energy metabolism. Although not supporting high-frequency spiking, fatty acid β-oxidation in mitochondria and oligodendroglial peroxisomes protects axons from conduction blocks when glucose is limiting. Disruption of the glucose transporter GLUT1 expression in oligodendrocytes of adult mice perturbs myelin homeostasis in vivo and causes gradual demyelination without behavioral signs. This further suggests that the imbalance of myelin synthesis and degradation can underlie myelin thinning in aging and disease., (© 2024. The Author(s).)

ConspectusIn recent years, the controlled assembly/disassembly of exogenous chemical components inside cells has become an emerging approach to regulating cell functions. However, the construction of dynamic material chemistry systems in living cells always remains highly challenging due to the complicated intracellular microenvironment. Nucleic acid is a category of biological components that can achieve efficient molecular assembly via specific base-pairing and perform biological functions in the intracellular microenvironment. Deoxyribonucleic acid (DNA) molecules exhibit the superior performance of intracellular assembly, including sequence programmability, molecule recognition ability, and nanostructure predictability, as well as the unique biological functions that traditional synthetic polymers do not carry, showing great superiority in the construction of dynamic material chemistry systems. Moreover, the technologies of DNA synthesis are relatively mature, and the conjugation of DNA with functional small molecules can be achieved through established chemical synthesis methods, facilitating the construction of DNA-based dynamic materials with more functions. In addition, a few specific DNA molecules have been proven to show responsiveness toward different stimuli, functioning as dynamic modules.In this Account, we summarize our recent work in dynamic chemistry of DNA-based nanoassemblies in living cells from the perspective of stimulus types including enzyme, H + , glutathione (GSH), adenosine triphosphate (ATP), and light. Upon the specific stimuli, DNA-based nanoassemblies undergo precise assembly in living cells, executing disassembly or aggregation, which consequently affects the functions and behaviors of living cells. In the first part, we describe the interactions between DNA-based nanoassemblies and intracellular enzymes, namely the enzymatic cleavage of intracellular enzymes on the DNA or RNA sequences. In the second part, we summarize the effects of H + in lysosomes on DNA-based nanoassemblies, including the formation of a tetraplex i-motif structure and the decomposition of acid-degradable polymeric coating. In the third part, we discuss the mechanism of GSH responsiveness of DNA-based nanoassemblies, including the breaking of disulfide bonds and reduction-responsive nanoparticles. In the fourth part, we describe the ATP-mediated conformational transition for the specific release of functional RNA sequences. In the fifth part, we demonstrate the light-mediated spatiotemporally dynamic chemistry of DNA-based nanoassemblies. In summary, based on the achievements of our group in the study of dynamic chemistry of DNA-based nanoassemblies, the assembly, disassembly, and reassembly in living cells are well-controlled, the regulation of cellular functions are explored, and the new strategies for cancer therapeutics are demonstrated. We envision that our work on the dynamic chemistry of DNA-based nanoassembly is a new paradigm for constructing dynamic material chemistry systems inside living cells, and will facilitate the development of precision medicine.

Research Question: Granulosa cells (GCs) dysfunction plays a crucial role in the pathogenesis of polycystic ovary syndrome (PCOS). It is reported that YTH domain-containing family protein 2 (YTHDF2) is upregulated in mural GCs of PCOS patients. What effect does the differential expression of YTHDF2 have in PCOS patients?, Design: Mural GCs and cumulus GCs from 15 patients with PCOS and 15 ovulatory controls and 4 cases of pathological sections in each group were collected. Real-time PCR, Western Blot, immunohistochemistry, and immunofluorescence experiments were conducted to detect gene and protein expression. RNA immunoprecipitation assay was performed to evaluate the binding relationship between YTHDF2 and MSS51. Mitochondrial morphology, cellular ATP and ROS levels and glycolysis-related gene expression were detected after YTHDF2 overexpression or MSS51 inhibition., Results: In the present study, we found that YTHDF2 was upregulated in GCs of PCOS patients while MSS51 was downregulated. YTHDF2 protein can bind to MSS51 mRNA and affect MSS51 expression. The reduction of MSS51 expression or the increase in YTHDF2 expression can lead to mitochondrial damage, reduced ATP levels, increased ROS levels and reduced expression of LDHA, PFKP and PKM., Conclusions: YTHDF2 may regulate the expression of MSS51, affecting the structure and function of mitochondria in GCs and interfering with cellular glycolysis, which may disturb the normal biological processes of GCs and follicle development in PCOS patients., Competing Interests: Declaration of competing interest All authors have declared that they have no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Objective: This study aimed to clarify the difference in Fusobacterium nucleatum (F. nucleatum) induced inflammatory cytokines and nod-like receptor protein 3 (NLRP3) inflammasomes dysregulation among three periodontal cells., Methods: Oral epithelial cells (HIOECs), THP-1 macrophages, and human gingival fibroblasts (HGFs) were exposed to F. nucleatum with/without adenosine triphosphate (ATP) and nigericin (Nig). Cell morphology was assessed by scanning electron microscopy. qRT-PCR, protein microarrays, and bioinformatic methods were used to evaluate the cytokines and their complex interplay. NLRP3 inflammasomes activation was detected by western blotting and ELISA., Results: F. nucleatum adhered to and invaded cells. In HIOECs, F. nucleatum enhanced interleukin (IL)-1α/1β/6/10/13, TNF-α, and interferon (IFN)-γ expression. In THP-1 macrophages, F. nucleatum up-regulated IL-1α/1β/6/10 and TNF-α levels. In HGFs, F. nucleatum increased IL-6 levels. F. nucleatum and ATP synergistically boosted IFN-γ level in THP-1 macrophages and IL-13 level in HGFs. IL-1α/1β/6, and TNF-α served as epicenters of the inflammatory response. Additionally, F. nucleatum activated NLRP3 inflammasomes in HIOECs, and ATP/Nig boosted the activation. F. nucleatum also triggered NLRP3 inflammasomes in THP-1 macrophages, but in HGFs, only NLRP3 and caspase-1 levels were elevated., Conclusion: F. nucleatum infiltrated periodontal supporting cells and dysregulated inflammatory cytokines and NLRP3 inflammasomes., (© 2024 Wiley Periodicals LLC.)

The interplay between purinergic receptors as well as pattern recognition receptors like Toll-like receptors (TLRs) and NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) might have a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to determine and compare the concentrations of the damage-associated molecular patterns (DAMPs) heat shock protein 70 (Hsp70) and adenosine triphosphate (ATP), and gene expression of their respective receptors as well as NLRP3 inflammasome-related molecules in the peripheral blood of patients with end-stage COPD before and 1 year after lung transplantation (LT). Lung function was assessed by spirometry and diffusion capacity for carbon monoxide (DLCO). Quantitative polymerase chain reaction (qPCR) was applied for detection of TLR2, TLR4, P2X7R, P2Y2R, IL1B, CASP1, and NLRP3 expression. High-sensitivity ELISA kits were used for extracellular (e) Hsp70 and IL-1β, and luminescence assay for eATP measurements. Concentrations of eHsp70 and eATP as well as IL-1β were significantly increased in the plasma of end-stage COPD patients and significantly decreased after LT. In addition, TLR4, P2Y2R, IL1B, CASP1, and NLRP3 expression was up-regulated in COPD patients before LT, while it was significantly suppressed after LT. In conclusion, it could be assumed that NLRP3 inflammasome is activated in the peripheral blood of end-stage COPD patients and that eHsp70 and eATP could be responsible for its activation through triggering their receptors. On the other hand, previously enhanced pro-inflammatory reactions seem to be suppressed to the healthy population levels in lung recipients without allograft rejection., Competing Interests: Declarations Ethics approval The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of University Hospital Centre Zagreb and the Ethical Committee for Experimentation of Faculty of Pharmacy and Biochemistry, University of Zagreb (Zagreb, Croatia); (Approval Protocol Numbers: 02/21/JG and 251-62-03-14-78, respectively). Consent to participate Informed consent was obtained from all subjects involved in the study. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Rapid propagation of tumor cells requires plenty of energy, which is adenosine triphosphate (ATP) dependent. ATP inhibition in tumors not only results in the starvation of tumor cells but also down-regulation of the level of heat shock proteins (HSPs), which usually increase during traditional photothermal therapy (PTT), especially when the temperature is up 50 °C. 2-deoxy-D-glucose (2DG) is an anti-glycolytic reagent and can be used as an efficient agent for ATP inhibition in tumors. Compared with typical PTT, low-temperature mild photothermal therapy (MPTT) is receiving more and more attention because it avoids the high temperatures causing damage to the normal tissue, and the increase of HSPs which decrease PTT. Here, multifunctional polypeptide nanoparticles pDG@Ahx conjugating both a NIR probe Ahx-BDP and 2DG into the side chain of the amphiphilic polypeptide have been prepared. In vitro and in vivo studies reveal that the as-prepared nanoparticles achieve a synergistic effect of starvation/MPTT/PDT (photodynamic therapy), and it provides a new strategy to NIR-I/II fluorescence imaging-guided starvation/MPTT/PDT synergy therapy for tumors., (© 2024 Wiley‐VCH GmbH.)

Molecular chaperone heat shock protein 90 (Hsp90) is a ubiquitous regulator that fine-tunes and remodels diverse client proteins, exerting profound effects on normal biology and diseases. Unraveling the mechanistic details of Hsp90's function requires atomic-level insights into its client interactions throughout the adenosine triphosphate-coupled functional cycle. However, the structural details of the initial encounter complex in the chaperone cycle, wherein Hsp90 adopts an open conformation while engaging with the client, remain elusive. Here, using nuclear magnetic resonance spectroscopy, we determined the solution structure of Hsp90 in its open state, bound to a disordered client. Our findings reveal that Hsp90 uses two distinct binding sites, collaborating synergistically to capture discrete hydrophobic segments within client proteins. This bipartite interaction generates a versatile complex that facilitates rapid conformational sampling. Moreover, our investigations spanning various clients and Hsp90 orthologs demonstrate a pervasive mechanism used by Hsp90 orthologs to accommodate the vast array of client proteins. Collectively, our work contributes to establish a unified conceptual and mechanistic framework, elucidating the intricate interplay between Hsp90 and its clients., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

It is predicted that ultra-short electric field pulses (nanosecond) can selectively permeabilize intracellular structures (e.g., mitochondria) without significant effects on the outer cell plasma membrane. Such a phenomenon would have high applicability in cancer treatment and could be employed to modulate cell death type or immunogenic response. Therefore, in this study, we compare the effects of 100 µs x 8 pulses (ESOPE - European Standard Operating Procedures on Electrochemotherapy) and bursts of 100 ns pulses for modulation of the mitochondria membrane potential. We characterize the efficacies of various protocols to trigger permeabilization, depolarize mitochondria (evaluated 1 h  after treatment), the extent of ATP depletion and generation of reactive oxygen species (ROS). Finally, we employ the most prominent protocols in the context of Ca 2+ electrochemotherapy in vitro. We provide experimental proof that 7.5-12.5 kV/cm x 100 ns pulses can be used to modulate mitochondrial potential, however, the permeabilization of the outer membrane is still a prerequisite for depolarization. Similar to 100 µs x 8 pulses, the higher the permeabilization rate, the higher the mitochondrial depolarization. Nevertheless, 100 ns pulses result in lesser ROS generation when compared to ESOPE, even when the energy input is several-fold higher than for the microsecond procedure. At the same time, it shows that even the short 100 ns pulses can be successfully used for Ca 2+ electrochemotherapy, ensuring excellent cytotoxic efficacy., 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 B.V. All rights reserved.)

External stress disrupts the balance of protein homeostasis, necessitating the involvement of heat shock proteins (Hsps) in restoring equilibrium and ensuring cellular survival. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius, lacks the conventional Hsp100, Hsp90, and Hsp70, relying solely on a single ATP-dependent Group II chaperonin, Hsp60, comprising three distinct subunits (α, β, and γ) to refold unfolded substrates and maintain protein homeostasis. Hsp60 forms three different complexes, namely Hsp60αβγ, Hsp60αβ, and Hsp60β, at temperatures of 60 °C, 75 °C, and 90 °C, respectively. This study delves into the intricacies of Hsp60 complexes in S. acidocaldarius, uncovering their ability to form oligomeric structures in the presence of ATP. The recognition of substrates by Hsp60 involves hydrophobic interactions, and the subsequent refolding process occurs in an ATP-dependent manner through charge-driven interactions. Furthermore, the Hsp60β homo-oligomeric complex can protect the archaeal and eukaryotic membrane from stress-induced damage. Hsp60 demonstrates nested cooperativity in ATP hydrolysis activity, where MWC-type cooperativity is nested within KNF-type cooperativity. Remarkably, during ATP hydrolysis, Hsp60β, and Hsp60αβ complexes exhibit a mosaic behavior, aligning with characteristics observed in both Group I and Group II chaperonins, adding a layer of complexity to their functionality., (© 2024 Federation of European Biochemical Societies.)

Viruses compete with each other for limited cellular resources, and some deliver defence mechanisms that protect the host from competing genetic parasites 1 . The phage antirestriction induced system (PARIS) is a defence system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB) 2 . However, the mechanism by which AriA and AriB function in phage defence is unknown. Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-electron microscopy to determine the structure of this complex, thereby explaining how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving host lysine transfer RNA. Phage T5 subverts PARIS immunity through expression of a lysine transfer RNA variant that is not cleaved by PARIS, thereby restoring viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids 3 ., (© 2024. The Author(s).)

Autonomous generation of energy, specifically adenosine triphosphate (ATP), is critical for sustaining the engineered functionalities of synthetic cells constructed from the bottom-up. In this mini-review, we categorize studies on ATP-producing synthetic cells into three different approaches: photosynthetic mechanisms, mitochondrial respiration mimicry, and utilization of non-conventional approaches such as exploiting synthetic metabolic pathways. Within this framework, we evaluate the strengths and limitations of each approach and provide directions for future research endeavors. We also introduce a concept of building ATP-generating synthetic organelle that will enable us to mimic cellular respiration in a simpler way than current strategies. This review aims to highlight the importance of energy self-production in synthetic cells, providing suggestions and ideas that may help overcome some longstanding challenges in this field., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Acute kidney injury (AKI) is a critical health issue with high mortality and morbidity rates in hospitalized individuals. The complex pathophysiology and underlying health conditions further complicate AKI management. Growing evidence suggests the pivotal role of ion channels in AKI progression, through promoting tubular cell death and altering immune cell functions. Among these channels, P2X purinergic receptors emerge as key players in AKI pathophysiology. P2X receptors gated by adenosine triphosphate (ATP), exhibit increased extracellular levels of ATP during AKI episodes. More importantly, certain P2X receptor subtypes upon activation exacerbate the situation by promoting the release of extracellular ATP. While therapeutic investigations have primarily focused on P2X 4 and P2X 7 subtypes in the context of AKI, while understanding about other subtypes still remains limited. Whilst some P2X antagonists show promising results against different types of kidney diseases, their role in managing AKI remains unexplored. Henceforth, understanding the intricate interplay between P2X receptors and AKI is crucial for developing targeted interventions. This review elucidates the functional alterations of all P2X receptors during normal kidney function and AKI, offering insights into their involvement in AKI. Notably, we have highlighted the current knowledge of P2X receptor antagonists and the possibilities to use them against AKI in the future. Furthermore, the review delves into the pathways influenced by activated P2X receptors during AKI, presenting potential targets for future therapeutic interventions against this critical condition., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

GroEL, a chaperone protein responsible for peptide and denatured protein folding, undergoes substantial conformational changes driven by ATP binding and hydrolysis during folding. Utilizing these conformational changes, we demonstrated the GroEL-mediated regioselective photocyclodimerization of 2-anthracenecarboxylic acid (AC) using ATP hydrolysis as an external stimulus. We designed and prepared an optimal GroEL mutant to employ in a docking simulation that has been actively used in recent years. Based on the large difference in the motif of hydrogen bonds between AC and GroEL mutant compared with the wild-type, we predicted that GroEL MEL , in which the 307‒309th amino acid residues were mutated to Ala, could alter the orientation of bound AC in GroEL. The GroEL MEL -mediated photocyclodimerization of AC can be used for regioselective inversion upon ATP addition to a moderate extent., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Metastasis is the primary stumbling block to the treatment of bladder cancer (BC). In order to spread, tumor cells must acquire increased migratory and invasive capacity, which is tightly linked with pseudopodia formation. Here, we unravel the effects of sulforaphane (SFN), an isothiocyanate in cruciferous vegetables, on the assembly of pseudopodia and BC metastasis, and its molecular mechanism in the process. Our database analysis revealed that in bladder tumor, pseudopodia-associated genes, CTTN, WASL and ACTR2/ARP2 are upregulated. SFN caused lamellipodia to collapse in BC cells by blocking the CTTN-ARP2 axis. SFN inhibited invadopodia formation and cell invasion by reducing WASL in different invasive BC cell lines. The production of ATP, essential for the assembly of pseudopodia, was significantly increased in bladder tumors and strongly inhibited by SFN. Overexpressing AKT1 reversed the downregulation of ATP in SFN-treated bladder cancer cells and restored filopodia and lamellipodia morphology and function. Bioluminescent imaging showed that SFN suppressed BC metastases to the lung of nude mice while downregulating Cttn and Arp2 expression. Our study thus reveals mechanisms of SFN action in inhibiting pseudopodia formation and highlights potential targeting options for the therapy of metastatic bladder cancer., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β 2 and α 2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β 2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β 2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

The limitations of self-assembled polymeric nanoparticles for cancer therapy, including instability in the bloodstream, non-specific targeting of cancer cells, and unregulated intracellular drug delivery, were effectively addressed by the development of core-shell SNX@PLL-FPBA/mHA NPs. The core was SNX@PLL-FPBA NPs prepared from polylysine conjugated 3-fluoro-4-carboxyphenylboronic acid (PLL-FPBA) self-assembly and SNX encapsulation, while the shell was methacrylate-modified hyaluronic acid (mHA) adhering to the core by electrostatic interactions and subsequently stabilized by photo-crosslinking, without the use of any organic solvent. SNX@PLL-FPBA/mHA NPs exhibited good stability in varying ionic strengths (0-0.30 M NaCl), pH levels (6.8 and 7.4), and plasma environments mimicking the blood, ensuring their efficacy in systemic circulation. The drug delivery from the nanoparticles was highly sensitive to ATP/Hyals stimuli (82 % within 48 h), closely mimicking the intracellular environment of breast cancer cells. The nanoparticles demonstrated good hemocompatibility and non-toxicity towards human skin fibroblasts. Efficient internalization of SNX@PLL-FPBA/mHA NPs by MCF-7 and MDA-MB-231 breast cancer cells was observed by CLSM and flow cytometry. The intracellular ATP/Hyals stimuli triggered the rapid drug delivery and induced cellular apoptosis. Thus, SNX@PLL-FPBA/mHA NPs were a promising drug nanocarrier for breast cancer therapy, offering improved stability, targeted delivery, and controlled drug release to enhance treatment outcomes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Preyssler-type polyoxotungstate ({P 5 W 30 }) belongs to the family of polyanionic metal-oxides formed by group V and VI metal ions, such as V, Mo and W, commonly known as polyoxometalates (POMs). POMs have demonstrated inhibitory effect on a significant number of ATP-binding proteins in vitro. Purinergic P2 receptors, widely expressed in eukaryotic cells, contain extracellularly oriented ATP-binding sites and play many biological roles with health implications. In this work, we use the immortalized mouse hippocampal neuronal HT-22 cells in culture to study the effects of {P 5 W 30 } on the cytosolic Ca 2+ concentration. Changes in cytosolic Ca 2+ concentration were monitored using fluorescence microscopy of HT-22 cells loaded with the fluorescent Ca 2+ indicator Fluo3. 31 P-Nuclear magnetic resonance measurements of {P 5 W 30 } indicate its stability in the medium used for cytosolic Ca 2+ measurements for over 30 min. The findings reveal that addition of {P 5 W 30 } to the extracellular medium induces a sustained increase of the cytosolic Ca 2+ concentration within minutes. This Ca 2+ increase is triggered by extracellular Ca 2+ entry into the cells and is dose-dependent, with a half-of-effect concentration of 0.25 ± 0.05 μM {P 5 W 30 }. In addition, after the {P 5 W 30 }-induced cytosolic Ca 2+ increase, the transient Ca 2+ peak induced by extracellular ATP is reduced up to 100% with an apparent half-of-effect concentration of 0.15 ± 0.05 μM {P 5 W 30 }. Activation of metabotropic purinergic P2 receptors affords about 80% contribution to the increase of Fluo3 fluorescence elicited by {P 5 W 30 } in HT-22 cells, whereas ionotropic receptors contribute, at most, with 20%. These results suggest that {P 5 W 30 } could serve as a novel agonist of purinergic P2 receptors., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Annette Rompel, Nadiia I. Gumerova reports financial support was provided by Austrian Science Fund. Manuel Aureliano reports financial support was provided by Foundation for Science and Technology. Carlos Gutierrez-Merino, Ana M. Mata reports financial support was provided by Spanish State Research Agency. 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. Published by Elsevier Inc.)

Crassulacean acid metabolism (CAM) leaves are characterized by nocturnal acidification and diurnal deacidification processes related with the timed actions of phosphoenolpyruvate carboxylase and Rubisco, respectively. How CAM leaves manage cytosolic proton homeostasis, particularly when facing massive diurnal proton effluxes from the vacuole, remains unclear. A 12-phase flux balance analysis (FBA) model was constructed for a mature malic enzyme-type CAM mesophyll cell in order to predict diel kinetics of intracellular proton fluxes. The charge- and proton-balanced FBA model identified the mitochondrial phosphate carrier (PiC, Pi/H + symport), which provides Pi to the matrix to sustain ATP biosynthesis, as a major consumer of cytosolic protons during daytime (> 50%). The delivery of Pi to the mitochondrion, co-transported with protons, is required for oxidative phosphorylation and allows sufficient ATP to be synthesized to meet the high energy demand during CAM Phase III. Additionally, the model predicts that mitochondrial pyruvate originating from decarboxylation of malate is exclusively exported to the cytosol, probably via a pyruvate channel mechanism, to fuel gluconeogenesis. In this biochemical cycle, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acts as another important cytosolic proton consumer. Overall, our findings emphasize the importance of mitochondria in CAM and uncover a hitherto unappreciated role in metabolic proton homeostasis., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Background: As a part of the catecholamines, dopamine receptors (DRs) have not been extensively studied like β3-AR in the thermogenesis process. The present study investigates the effect of DRD5 in browning events and ATP-consuming futile cycles., Methods: siRNA technology, qPCR, immunoblot analysis, immunofluorescence, and staining methods were used to investigate the effect of DRD5 on 3T3-L1 and C2C12 cells., Results: si Ddr5 increased lipogenesis-associated effectors, and adipogenesis markers while reducing the expression of beige fat effectors. ATP-consuming futile cycle markers were also reduced following the si Drd5 . On the contrary, pharmacological activation of DRD5 stimulated these effectors. Our mechanistic studies elucidated that DRD5 mediates fat browning via the cAMP-PKA-p38 MAPK signalling pathway in 3T3-L1 cells as well as the cAMP-SERCA-RyR pathway for the ATP-consuming futile cycles in both cells., Conclusions: si Drd5 positively regulates browning and ATP-consuming futile cycles, and understanding its functions will provide insights into novel strategies to treat obesity.

This study used 16S rRNA gene sequencing and metatranscriptomic analysis to comprehensively illustrate how ammonia stress influenced medium-chain fatty acids (MCFA) biosynthesis. MCFA synthesis was inhibited at total ammonia nitrogen (TAN) concentrations above 1000 mg N/L. TAN stress hindered organic hydrolysis, acidification, and volatile fatty acids elongation. Chain-elongating bacteria (e.g., Clostridium_sensu_stricto_12, Clostridium_sensu_stricto_1, Caproiciproducens) abundance remained unchanged, but their activity decreased, partially due to the increased reactive oxygen species. Metatranscriptomic analysis revealed reduced activity of enzymes critical for MCFA production under TAN stress. Fatty acid biosynthesis pathway rather than reverse β-oxidation pathway primarily contributed to MCFA production, and was inhibited under TAN stress. Functional populations likely survived TAN stress through osmoprotectant generation and potassium uptake regulation to maintain osmotic pressure, with NADH-ubiquinone oxidoreductase potentially compensating for ATP loss. This study enhances understanding of MCFA biosynthesis under TAN stress, aiding MCFA production system stability and efficiency improvement., 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.)

The aim of our study was to investigate the effects of cyanobacterial metabolites: microcystin-LR (MC-LR) anabaenopeptin-A (ANA-A), cylindrospermopsin (CYL), their binary and ternary mixtures on rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1) cell line. We determined the following cell parameters: Hoechst and propidium iodide (PI) double staining, intracellular ATP level with luminometric assay, glutathione level with ThiolTracker Violet®- glutathione detection reagent and cytoskeletal F-actin fluorescence. The results showed that although reduction of Hoechst fluorescence was observed in both binary and ternary combinations of cyanobacterial metabolites, the mixture of MC-LR + ANA-A + CYL was the most potent inhibitor (EC50 = 148 nM). PI fluorescence and ATP levels were more increased in the cells exposed to the mixtures than those exposed to the individual metabolites with synergistic toxic changes suggesting apoptosis as the mechanism of cell death. Reduced glutathione level was also decreased in cells exposed both to single metabolites and their mixtures with the highest decrease and synergistic effects at 334 nM MC-LR+334 nM ANA-A+ 334 nM CYL suggesting induction oxidative stress by the tested compounds. Reduction of F-actin fluorescence was found in the cells from all of the groups exposed to individual metabolites and their mixtures, however the highest level of inhibition showed the binary MC-LR + CYL and the ternary MC-LR + ANA-A + CYL with synergistic interactions. The study suggests that in natural conditions fish gill cells may be very sensitive to individual cyanobacterial metabolites and more prone to their binary and ternary mixtures., 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.)

Mitochondrial creatine kinase (mtCK) regulates the "fast" export of phosphocreatine to support cytoplasmic phosphorylation of ADP to ATP which is more rapid than direct ATP export. Such "creatine-dependent" phosphate shuttling is attenuated in several muscles, including the heart, of the D2.mdx mouse model of Duchenne muscular dystrophy at only 4 weeks of age. However, the degree to which creatine-dependent and -independent systems of phosphate shuttling progressively worsen or potentially adapt in a hormetic manner throughout disease progression remains unknown. Here, we performed a series of proof-of-principle investigations designed to determine how phosphate shuttling pathways worsen or adapt in later disease stages in D2.mdx (12 months of age). We also determined whether changes in creatine-dependent phosphate shuttling are linked to alterations in mtCK thiol redox state. In permeabilized muscle fibres prepared from cardiac left ventricles, we found that 12-month-old male D2.mdx mice have reduced creatine-dependent pyruvate oxidation and elevated complex I-supported H 2 O 2 emission (mH 2 O 2 ). Surprisingly, creatine-independent ADP-stimulated respiration was increased and mH 2 O 2 was lowered suggesting that impairments in the faster mtCK-mediated phosphocreatine export system resulted in compensation of the alternative slower pathway of ATP export. The apparent impairments in mtCK-dependent bioenergetics occurred independent of mtCK protein content but were related to greater thiol oxidation of mtCK and a more oxidized cellular environment (lower GSH:GSSG). Next, we performed a proof-of-principle study to determine whether creatine-dependent bioenergetics could be enhanced through chronic administration of the mitochondrial-targeting, ROS-lowering tetrapeptide, SBT-20. We found that 12 weeks of daily treatment with SBT-20 (from day 4-∼12 weeks of age) increased respiration and lowered mH 2 O 2 only in the presence of creatine in D2.mdx mice without affecting calcium-induced mitochondrial permeability transition activity. In summary, creatine-dependent mitochondrial bioenergetics are attenuated in older D2.mdx mice in relation to mtCK thiol oxidation that seem to be countered by increased creatine-independent phosphate shuttling as a unique form of mitohormesis. Separate results demonstrate that creatine-dependent bioenergetics can also be enhanced with a ROS-lowering mitochondrial-targeting peptide. These results demonstrate a specific relationship between redox stress and mitochondrial hormetic reprogramming during dystrophin deficiency with proof-of-principle evidence that creatine-dependent bioenergetics could be modified with mitochondrial-targeting small peptide therapeutics., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare. Stealth Biotherapeutics supplied SBT-20 without funding., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Bacterial infections caused by multidrug-resistant (MDR) gram-negative strains carrying the mobile colistin resistance gene mcr-1 are serious threats to world public health due to the lack of effective treatments. Inhibition of the ATP synthase makes bacteria such as Staphylococcus aureus and Klebsiella pneumoniae more sensitive to polymyxin. This provides new strategies for treating infections caused by polymyxins-resistant bacteria carrying mcr-1. Six mcr-1-positive strains were isolated from clinical samples, and all were identified as Escherichia coli. Here we investigated several ATP synthase inhibitors, N,N'-dicyclohexylcarbodiimide (DCCD), resveratrol, and piceatannol, for their antibacterial effects against the mcr-1-positive strains combined with polymyxin B (POL). Checkerboard assay, time-kill assay, biofilm inhibition and eradication assay indicated the significant synergistic effect of ATP synthase inhibitors/POL combination in vitro. Meanwhile, mouse infection model experiment was also performed, showing a 5 log 10 reduction of the pathogen after treatment with the resveratrol/POL combination. Moreover, adding adenosine disodium triphosphate (Na 2 ATP) could inhibit the antibacterial effect of the ATP synthase inhibitors/POL combination. In conclusion, our study confirmed that inhibition of ATP production could increase the susceptibility of bacteria carrying mcr-1 to polymyxins. This provides a new strategy against polymyxins-resistant bacteria infection., (© 2024. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

AB598 is a CD39 inhibitory antibody being pursued for the treatment of solid tumors in combination with chemotherapy and immunotherapy. CD39 metabolizes extracellular adenosine triphosphate (eATP), an alarmin capable of promoting antitumor immune responses, into adenosine, an immuno-inhibitory metabolite. By inhibiting CD39, the consumption of eATP is reduced, resulting in a proinflammatory milieu in which eATP can activate myeloid cells to promote antitumor immunity. The preclinical characterization of AB598 provides a mechanistic rationale for combining AB598 with chemotherapy in the clinic. Chemotherapy can induce ATP release from tumor cells and, when preserved by AB598, both chemotherapy-induced eATP and exogenously added ATP promote the function of monocyte-derived dendritic cells via P2Y11 signaling. Inhibition of CD39 in the presence of ATP can promote inflammasome activation in in vitro-derived macrophages, an effect mediated by P2X7. In a MOLP8 murine xenograft model, AB598 results in full inhibition of intratumoral CD39 enzymatic activity, an increase in intratumoral ATP, a decrease of extracellular CD39 on tumor cells, and ultimately, control of tumor growth. In cynomolgus monkeys, systemic dosing of AB598 results in effective enzymatic inhibition in tissues, full peripheral and tissue target engagement, and a reduction in cell surface CD39 both in tissues and in the periphery. Taken together, these data support a promising therapeutic strategy of harnessing the eATP generated by standard-of-care chemotherapies to prime the tumor microenvironment for a productive antitumor immune response., (©2024 The Authors; Published by the American Association for Cancer Research.)

Human MRP1 protein plays a vital role in cancer multidrug resistance. Coumarins show promising pharmacological properties. Virtual screening, ADMET, molecular docking and molecular dynamics (MD) simulations were utilized as pharmacoinformatic tools to identify potential MRP1 inhibitors among coumarin derivatives. Using in silico ADMET, 50 hits were further investigated for their selectivity toward the nucleotide-binding domains (NBDs) of MRP1 using molecular docking. Accordingly, coumarin, its symmetrical ketone derivative Lig. No. 4, and Reversan were candidates for focused docking study with the NBDs domains compared with ATP. The result indicates that Lig. No. 4, with the best binding score, interacts with NBDs via hydrogen bonds with residues: GLN713, LYS684, GLY683, CYS682 in NBD1, and GLY1432, GLY771, SER769 and GLN1374 in NBD2, which mostly overlap with ATP binding residues. Moreover, doxorubicin (Doxo) was docked to the transmembrane domains (TMDs) active site of MRP1. Doxo interaction with TMDs was subjected to MD simulation in the NBDs free and occupied with Lig. No. 4 states. The results showed that Doxo interacts more strongly with TMD residues in inward facing feature of TMDs helices. However, when Lig. No. 4 exists in NBDs, Doxo interactions are different, and TMD helices show more outward-facing conformation. This result may suggest a partial competitive inhibition mechanism for the Lig. No. 4 on MRP1 compared with ATP. So, it may inhibit active complex formation by interfering with ATP entrance to NBDs and locking MRP1 conformation in outward-facing mode. This study suggests a valuable coumarin derivative that can be further investigated for potent MRP1 inhibitors.Communicated by Ramaswamy H. Sarma.

Ethylene oxide (E.O) is an epoxide compound, and it has been utilized as a sterilizer or production of ether compounds in several industries. Although the toxic effects of E.O on bacteria and mammals have been reported, its effects on male reproductive toxicity during sperm capacitation are not fully understood. Therefore, this study was designed to evaluate the effects of E.O exposure during sperm capacitation. Boar spermatozoa were treated with various E.O concentrations (0, 0.1, 1, 10, and 100 μМ). After exposure, sperm motility, motion kinematics, capacitation status, intracellular ATP levels, cell viability, expression levels of protein kinase A (PKA) activation, and tyrosine phosphorylation were evaluated. Results revealed that E.O exposure significantly decreased sperm motility, motion kinematics, and intracellular ATP levels but significantly increased the capacitated spermatozoa. In addition, the PKA activation and tyrosine phosphorylation were abnormally changed. According to our results, E.O may cause toxic effects on sperm function during capacitation, which induces male reproductive toxicity. Consequently, we suggest that male reproductive toxicity should be considered when using E.O., 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.)

Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with β-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding β-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the β-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to β-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides., (© 2024. The Author(s).)

In recent years, overuse of antibiotics has led to emerging antibiotic-resistant strains of bacteria. Consequently, creating new, highly productive antibacterial agents is crucial. In this work, we synthesized copper-aluminum-zinc layered double hydroxide (Co-Al-Zn LDH) and modified it using adenosine triphosphate. After characterization, the enzyme-like activity of the prepared particles was evaluated. The results indicated peroxidase-mimic performance of ATP/Co-Al-Zn LDH with K m values of 0.38 mM and 1.69 mM for TMB (3,3',5,5'-tetramethylbenzidine) and hydrogen peroxide (H 2 O 2 ), respectively, which were lower than that of horseradish peroxidase. The highest peroxidase-like activity of ATP/Co-Al-Zn LDH was achieved at 20 °C, pH 4, with a 1.02 mg/mL catalyst, 231 μM TMB, and 1.9 mM H 2 O 2 . The bactericidal activity of the developed nanozyme was studied against E. coli and S. aureus. The peroxidase-mimic nanozyme decomposes H 2 O 2 and generates free radicals to kill bacteria in vitro. The minimum inhibitory concentration (MIC) of ATP/Co-Al-Zn LDH was 15 μg/mL and 20 μg/mL for S. aureus and E. coli, respectively. The morphological characteristics of the nanozyme-treated bacterial cells showed dramatic changes in bacterial morphology. Our results revealed higher antibacterial activity of ATP/Co-Al-Zn LDH against S. aureus. Therefore, the developed nanozyme could serve as a substitute for conventional antibiotics., Competing Interests: Declaration of competing interest We have no conflict of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Panax notoginseng (Burk.) F.H. Chen is the most traditional hemostatic herb in China. Our previous research found that 20(S)-protopanaxadiol showed the hemostatic effect. And 20(S)-panaxadiol (PD) has a similar structure to 20(S)-protopanaxadiol with a dammarane skeleton. So, this article mainly studies the hemostatic effect of PD. The mouse tail amputation and liver scratch models were used to detect the hemostatic effect of PD. Blood routine and plasma coagulation parameters were measured by using a blood analyzer. The platelet aggregometer analyzed the platelet aggregation rate and adenosine triphosphate (ATP) concentration. Moreover, the intracellular calcium concentration ([Ca2+]i), P-selectin (CD62P), PAC-1 (GP IIb/IIIa receptor marker), and cyclic adenosine monophosphate (cAMP) of platelets were also detected. The results showed that PD obviously shortened the bleeding time of the model mouse, affected the RBC and PLT parameters of rats, reduced APTT and TT, elevated FIB concentration, and promoted human/rat-washed platelet aggregation in vitro. PD promoted the release of ATP and [Ca2+]i and slightly increased the expression of CD62P and PAC-1 of platelets without 1 mM Ca2+. After adding 1 mM Ca2+, PD obviously increased ATP releasing and CD62P and GP IIb/IIIa expression rate and decreased the cAMP level of platelets. These parameter changes of PD-caused platelet were inhibited by vorapaxar. Besides, PD increased the phosphorylation of phosphoinositide 3-kinase/protein kinase B/glycogen synthase kinase 3β (PI3K/Akt/GSK3β) of human platelets. PD is an important hemostatic ingredient in Panax notoginseng, which induced platelet aggregation by affecting the calcium signaling and activating the PI3K/Akt/GSK3β signaling pathway. [ABSTRACT FROM AUTHOR]

Brown and beige adipocytes dissipate energy in a nonshivering thermogenesis manner, exerting beneficial effects on metabolic homeostasis. CHCHD10 is a nuclear-encoded mitochondrial protein involved in cristae organization; however, its role in thermogenic adipocytes remains unknown. We identify CHCHD10 as a novel regulator for adipocyte thermogenesis. CHCHD10 is dramatically upregulated during thermogenic adipocyte activation by PPARγ-PGC1α and positively correlated with UCP1 expression in adipose tissues from humans and mice. We generated adipocyte-specific Chchd10 knockout mice (Chchd10-AKO) and found that depleting CHCHD10 leads to impaired UCP1-dependent thermogenesis and energy expenditure in the fasting state, with no effect in the fed state. Lipolysis in adipocytes is disrupted by CHCHD10 deficiency, while augmented lipolysis through ATGL overexpression recovers adipocyte thermogenesis in Chchd10-AKO mice. Consistently, overexpression of Chchd10 activates thermogenic adipocytes. Mechanistically, CHCHD10 deficiency results in the disorganization of mitochondrial cristae, leading to impairment of oxidative phosphorylation complex assembly in mitochondria, which in turn inhibits ATP generation. Decreased ATP results in downregulation of lipolysis by reducing nascent protein synthesis of ATGL, thereby suppressing adipocyte thermogenesis. As a result, Chchd10-AKO mice are prone to develop high-fat diet-induced metabolic disorders. Together, our findings reveal an essential role of CHCHD10 in regulating lipolysis and the thermogenic program in adipocytes. [ABSTRACT FROM AUTHOR]

Objective To identify the role of fatty acid binding protein 3 (FABP3) in vascular fibrosis in Takayasu's arteritis (TAK) and to explore the underlying molecular mechanism. Methods The expression of FABP3 and extracellular matrix proteins (ECMs) were detected in aorta tissues from TAK patients (n  = 12) and healthy controls (n  = 8) by immunohistochemistry. The concentration of serum proteins was determined by ELISA. CCK8 and Ki67 staining were used to measure aorta adventitial fibroblast (AAF) proliferation. Widely targeted lipidomic profiling was used to screen for associated metabolic pathways. Changes in ECMs and fatty acid oxidation (FAO)-related enzymes were determined by RT-qPCR and Western blot. The interactions between FABP3 and these enzymes were explored with a co-immunoprecipitation (Co-IP) assay. Results The expression of FABP3 was increased in the thickened adventitia of TAK patients and was positively correlated with the serum expression of ECMs. FABP3 knockdown inhibited AAF proliferation and ECM production, whereas FABP3 overexpression enhanced these processes. Further analysis revealed that FABP3 upregulation promoted carnitine palmitoyltransferase 1A and carnitine/acylcarnitine carrier protein (CACT) expression, two key enzymes in FAO, as well as adenosine triphosphate (ATP) levels. FABP3 and CACT were co-localized in the adventitia and bound to each other in AAFs. Etomoxir reversed the enhanced FAO, ATP production, AAF proliferation and ECM production mediated by FABP3 upregulation. Treatment with 60 g/day curcumin granules for 3 months reduced the level of serum FABP3. Curcumin also inhibited vascular fibrosis by reducing FABP3-enhanced FAO in AAFs. Conclusion Elevated FABP3 expression accelerated vascular fibrosis in TAK, which was likely mediated by promoting FAO in AAFs. [ABSTRACT FROM AUTHOR]

Simple Summary: Drug repurposing has been effective for discovering novel treatments for cancer. The antipsychotic agent penfluridol was reported to suppress lung cancer growth via ATP energy deprivation. The aim of our study was to investigate how penfluridol influences energy metabolism in lung cancer cells. We observed that penfluridol inhibited mitochondrial oxidative phosphorylation (OXPHOS), but induced glycolysis to compensate for the loss of ATP caused by suppression of mitochondrial OXPHOS. We also confirmed that inhibition of glycolysis by 2-deoxy-D-glucose (2DG) significantly augmented the antitumor effects caused by penfluridol in vitro and in vivo. Our studies provide novel insights into repurposing penfluridol combined with 2-DG for lung cancer treatment. Energy metabolism is the basis for cell growth, and cancer cells in particular, are more energy-dependent cells because of rapid cell proliferation. Previously, we found that penfluridol, an antipsychotic drug, has the ability to trigger cell growth inhibition of lung cancer cells via inducing ATP energy deprivation. The toxic effect of penfluridol is related to energy metabolism, but the underlying mechanisms remain unclear. Herein, we discovered that treatment of A549 and HCC827 lung cancer cells with penfluridol caused a decrease in the total amount of ATP, especially in A549 cells. An Agilent Seahorse ATP real-time rate assay revealed that ATP production rates from mitochondrial respiration and glycolysis were, respectively, decreased and increased after penfluridol treatment. Moreover, the amount and membrane integrity of mitochondria decreased, but glycolysis-related proteins increased after penfluridol treatment. Furthermore, we observed that suppression of glycolysis by reducing glucose supplementation or using 2-deoxy-D-glucose (2DG) synergistically enhanced the inhibitory effect of penfluridol on cancer cell growth and the total amount of mitochondria. A mechanistic study showed that the penfluridol-mediated energy reduction was due to inhibition of critical regulators of mitochondrial biogenesis, the sirtuin 1 (SIRT1)/peroxisome-proliferator-activated receptor co-activator-1α (PGC-1α) axis. Upregulation of the SIRT1/PGC-1α axis reversed the inhibitory effect of penfluridol on mitochondrial biogenesis and cell viability. Clinical lung cancer samples revealed a positive correlation between PGC-1α (PPARGC1A) and SIRT1 expression. In an orthotopic lung cancer mouse model, the anticancer activities of penfluridol, including growth and metastasis inhibition, were also enhanced by combined treatment with 2DG. Our study results strongly support that a combination of repurposing penfluridol and a glycolysis inhibitor would be a good strategy for enhancing the anticancer activities of penfluridol in lung cancer. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the energy pathway contributions and physiological and performance responses between a 10 × 6-second intermittent sprint test (IST) and a 60-second single maximal test (SMT). Seventeen highly trained male Greco-Roman wrestlers participated in this study. Participants completed the 60-second upper-body Wingate tests, both intermittent and single forms. The contributions of the oxidative, glycolytic, and ATP-PCr pathways were estimated using mathematical methods based on lactate values and oxygen consumption kinetics of rest, exercise, and recovery phases. The main findings indicated that total energy expenditure (TEE) and the contribution of oxidative, glycolytic, and ATP-PCr pathways were 514 kJ, 45%, 11%, and 44% for IST (overall: sprints + rest intervals); 333 kJ, 14%, 17%, and 69% for IST (sprints only); and 159 kJ, 31%, 38%, and 31% for SMT, respectively. TEE and ATP-PCR pathway contributions were higher in the IST (both overall and sprint only), whereas glycolytic pathway contribution and delta lactate were higher in the SMT. Absolute oxidative contribution was similar, but relative oxidative contribution was higher in the SMT. Additionally, mean power was higher in the IST than SMT, whereas peak power, peak and mean heart rate, and ratings of perceived exertion were similar. [ABSTRACT FROM AUTHOR]