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In this paper, we present a refined version of the previously proposed suspension contractile model based on catch muscle proteins of the Gray's mussel (Crenomytilus grayanus). The objective of this model was to test the current hypotheses about the catch state, a unique phenomenon observed in the adductor muscle of bivalve molluscs. This state allows the muscle to maintain the force developed by contraction for a long time with minimum energy expenditure. Our study has resolved the issue of constructing a catch muscle model capable of controlled contraction and relaxation in an ATP-resistant manner. As a result, we have gained insight into the functional role of myorod, a unique protein of the catch muscle, and have tested the modified myorod-links hypothesis of the catch state. Myorod was shown capable of forming strong and functional cross-links between actin and synthetic thick filaments. This finding removes the last shortcoming of the myorod-links hypothesis of the catch state. There is now no doubt that myorod plays the main role of the catch-link we have been seeking all this time., 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.)

Extracellular ATP (eATP) orchestrates vital processes in plants, akin to its role in animals. P2K1 is a crucial receptor mediating eATP effects. Immunoprecipitation tandem mass spectrometry data highlighted FERONIA's significant interaction with P2K1, driving us to explore its role in eATP signaling. Here, we investigated putative P2K1-interactor, FERONIA, which is a versatile receptor kinase pivotal in growth and stress responses. We employed a FERONIA loss-of-function mutant, fer-4 , to dissect its effects on eATP signaling. Interestingly, fer-4 showed distinct calcium responses compared to wild type, while eATP-responsive genes were constitutively upregulated in fer-4 . Additionally, fer-4 displayed insensitivity to eATP-regulated root growth and reduced cell wall accumulation. Together, these results uncover a role for FERONIA in regulating eATP signaling. Overall, our study deepens our understanding of eATP signaling, revealing the intricate interplay between P2K1 and FERONIA impacting the interface between growth and defense.

An ultrasensitive strategy for in-situ visual monitoring of ATP in a single living tumor cell during mitochondria-targeted photodynamic therapy (PDT) process with high spatiotemporal resolution was proposed using surface-enhanced Raman scattering (SERS) 3D imaging technique. The nanostructures consisting of Au-Ag 2 S Janus nanoparticles functionalized with both Au nanoparticles linked by a DNA chain and a mitochondrial-targeting peptide (JMDA NPs) were deliberately employed to target mitochondria. The JMDA NPs exhibit excellent SERS activity and remarkable antitumor activity. The quantization of ATP relies on the intensity of the SERS probes bonded to the DNA, which shows a strong correlation with the generated hot spot between the Janus and the Au. Consequently, spatiotemporally controlled monitoring of ATP in the mitochondria of single living cells during the PDT process was achieved. Additionally, the JMDA NPs demonstrated remarkable capability for mitochondria-targeted PDT, providing significant antitumor effects and superior therapeutic safety both in vitro and in vivo. Our work presents an effective JMDA NPs-based SERS imaging strategy for in-situ and real-time 3D visualization of intracellular ATP in living tumor cells during the mitochondria-targeted PDT process, which enables significant information on the time point of PDT treatment and is beneficial to precious PDT applications in tumor therapy., 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.)

Proteins must fold into their native structure to carry out cellular functions. However, they can sometimes misfold into non-native structures, leading to reduced efficiency or malfunction. Chaperones help prevent misfolding by guiding proteins to their active state using energy from ATP hydrolysis. Experiments have revealed numerous kinetic and structural aspects of how various chaperones facilitate the folding of proteins into their native structure. However, what remains missing is a fundamental theoretical understanding of their operational mechanisms, especially the limits and constraints imposed on their efficiency by energy flow and dissipation. To address this, we built a kinetic model of the Hsp70 chaperone system by incorporating all key structural and kinetic details. Then, using the chemical kinetic equations, we investigate how energy expenditure shapes the efficiency of Hsp70 chaperones in the proper folding of misfolded proteins. We show that ATP consumption by chaperones significantly enhances the folding of proteins into their native states. Our investigations reveal that a chaperone achieves optimal efficiency when its binding to misfolded proteins is much faster than the misfolding kinetics of that protein. We also demonstrate the presence of an upper bound on a chaperone's efficiency of protein folding and its overall rescue rate. This upper bound increases with energy dissipation until it reaches a saturation point. Furthermore, we show a speed-energy-efficiency trade-off in chaperone action, demonstrating that it is impossible to simultaneously optimize the efficiency of chaperone-assisted protein folding and the energy efficiency of the process.

Binding multiple sites within proteins with bivalent compounds is a strategy for developing uniquely active agents. A new class of dual-site inhibitors has emerged targeting the epidermal growth factor receptor (EGFR) anchored to both the orthosteric (ATP) and allosteric sites. Despite proof-of-concept successes, enabling selectivity against oncogenic activating mutations has not been achieved and classifying these inhibitors among kinase inhibitors remains underexplored. This study investigates the structure-activity relationships, binding modes, and biological activity of ATP-allosteric bivalent inhibitors (AABIs). We find that AABIs selectively inhibit drug-resistant EGFR mutants (L858R/T790M and L858R/T790M/C797S) by anchoring a methyl isoindolinone moiety along the αC-helix channel of the allosteric site. In contrast, related Type I 1 / 2 inhibitors target wild-type EGFR but are less effective against resistant mutants. This shift in selectivity demonstrates that mutant-selective AABIs classify as "Type V" bivalent inhibitors.

Hsp90, a crucial molecular chaperone, regulates diverse client proteins, impacting both normal biology and disease. Central to its function is its conformational plasticity, driven by ATPase activity and client interactions. However, comprehensive insights into Hsp90's dynamic molecular transitions remain elusive. Using solution NMR spectroscopy, we reveal how ATP binding, hydrolysis, and client engagement drive conformational and dynamic shifts in E. coli Hsp90, HtpG, through its chaperone cycle. Pronounced conformational fluctuations occur, especially in regions crucial for nucleotide binding and conformational transitions. ATP binding induces slow-exchanging conformations, representing discrete on-path transition states from open to closed forms, while ATP hydrolysis shifts HtpG into a compact conformation. Client binding acts as an allosteric switch, dynamically priming HtpG for elevated chaperone activity and, therefore, its efficient remodeling. Here, we provide atomic-level insights into Hsp90's functional mechanism, highlighting the interplay of conformation, dynamics, nucleotide, and client interactions., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Resistance in pathogenic fungi necessitates the development of fungicides with new mechanisms of action. The Mps1 MAPK of Magnaporthe oryzae , the pathogen of rice blast disease, has been shown to be a molecular target for fungicide research. Here, we present compound TAK-733 that interacts with the common docking (CD) site of Mps1 and can be used in combination with ATP-competitive inhibitors. We initially identified compounds PLX-4720 and TAK-733 that interact with Mps1. Subsequent assays show that PLX-4720 is an ATP-competitive inhibitor, whereas TAK-733 binds to the CD site of Mps1─an interaction site for its MAPKK─but not to the ATP-binding pocket as it does in the kinase MEK1. In vivo assays demonstrated that TAK-733 exhibits combinational effects with ATP-competitive inhibitors PLX-4720 and A378-0. Collectively, we present TAK-733 as having a new mechanism of action suitable for combinational application with ATP-competitive inhibitors in the management of pathogenic fungi.

Cyanobacterial photosynthesis (to produce ATP and NADPH) might have played a pivotal role in the endosymbiotic evolution to chloroplast. However, rather than meeting the ATP requirements of the host cell, the modern-day land plant chloroplasts are suggested to utilize photosynthesized ATP predominantly for carbon assimilation. This is further highlighted by the fact that the plastidic ADP/ATP carrier translocases from land plants preferentially import ATP. Here, we investigate the preferences of plastidic ADP/ATP carrier translocases from key lineages of photosynthetic eukaryotes including red algae, glaucophytes, and land plants. Particularly, we observe that the cyanobacterial endosymbionts expressing plastidic ADP/ATP carrier translocases from red algae and glaucophyte are able to export ATP and support ATP dependent endosymbiosis, whereas those expressing ADP/ATP carrier translocases from land plants preferentially import ATP and are unable to support ATP dependent endosymbiosis. These data are consistent with a scenario where the ancestral plastids may have exported ATP to support the bioenergetic functions of the host cell., Competing Interests: Competing interests: Authors declare that they have no competing interests., (© 2024. The Author(s).)

In cells, adenosine triphosphate (ATP) and guanosine triphosphate (GTP) molecules typically form tricoordinated or bicoordinated ATP·Mg 2+ or GTP·Mg 2+ complexes with Mg 2+ ions and bind to proteins, participating in and regulating many important cellular functions. The accuracy of their force field parameters plays a crucial role in studying the function-related conformations of ATP·Mg 2+ or GTP·Mg 2+ using molecular dynamics (MD) simulations. The parameters developed based on the methyl triphosphate model in existing AMBER force fields cannot accurately describe the conformational distribution of tricoordinated or bicoordinated ATP·Mg 2+ or GTP·Mg 2+ complexes in solution. In this study, we develop force field parameters for the triphosphate group based on the new ribosyl triphosphate model, considering the dihedral coupling effect, accurate van der Waals (vdW) interactions, and the influence of strongly polarized charges on conformational balance. The new force fields can accurately describe the conformational balance of tricoordinated and bicoordinated ATP·Mg 2+ or GTP·Mg 2+ conformations in solution and can be applied to simulate biological systems containing ATP·Mg 2+ or GTP·Mg 2+ complexes.

Nicotinamide Adenine Dinucleotide Phosphate (NADPH) plays a vital role in regulating redox homeostasis and reductive biosynthesis. However, if exogenous NADPH can be transported across the plasma membrane has remained elusive. In this study, we present evidence supporting that NADPH can traverse the plasma membranes of cells through a mechanism mediated by the P2X7 receptor (P2X7R). Notably, we observed an augmentation of intracellular NADPH levels in cultured microglia upon exogenous NADPH supplementation in the presence of ATP. The P2X7R-mediated transmembrane transportation of NADPH was validated with P2X7R antagonists, including OX-ATP, BBG, and A-438079, or through P2X7 knockdown, which impeded NADPH transportation into cells. Conversely, overexpression of P2X7 resulted in an enhanced capacity for NADPH transport. Furthermore, transfection of hP2X7 demonstrated the ability to complement NADPH uptake in native HEK293 cells. Our findings provide evidence for the first time that NADPH is transported across the plasma membrane via a P2X7R-mediated pathway. Additionally, we propose an innovative avenue for modulating intracellular NADPH levels. This discovery holds promise for advancing our understanding of the role of NADPH in redox homeostasis and neuroinflammation., 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 Inc.)

Kinesin-1 is a crucial motor protein that drives the microtubule-based movement of organelles, vital for cellular function and health. Mostly studied at pH 6.9, it moves at approximately 800 nm/s, covers about 1 μm before detaching, and hydrolyzes one ATP per 8 nm step. Given that cellular pH is dynamic and alterations in pH have significant implications for disease, understanding how kinesin-1 functions across different pH levels is crucial. To explore this, we executed single-molecule motility assays paired with precise optical trapping techniques over a pH range of 5.5-9.8. Our results show a consistent positive relationship between increasing pH and the enhanced detachment (off rate) and speed of kinesin-1. Measurements of the nucleotide-dependent off rate show that kinesin-1 exhibits the highest rate of ATPase activity at alkaline pH, while it demonstrates the optimal number of ATP turnover and cargo translocation efficiency at the acidic pH. Physiological pH of 6.9 optimally balances the biophysical activity of kinesin-1, potentially allowing it to function effectively across a range of pH levels. These insights emphasize the crucial role of pH homeostasis in cellular function, highlighting its importance for the precise regulation of motor proteins and efficient intracellular transport.

IL-6 is an important cytokine involved in metabolic, immunological, and cell-fate responses. It is released upon stimulation by skeletal muscle cells through partially characterized mechanisms. In some cell types, IL-6 has been reported to activate a positive feedback loop involving endocytic vesicles, but evidence is mostly based on transcription and signal transduction mechanisms and is very scarce in muscle cells. Our aim was to directly demonstrate the presence of positive feedback in the ATP-induced release of IL-6 into the supernatant of human skeletal muscle cultures. The total release (production) of IL-6 was reduced for higher volumes of supernatant, when the secreted IL-6 molecules are more diluted, and enhanced when the supernatant volume was lower. In addition, secretion was impaired both by tocilizumab, a blocker of human IL-6 receptors, and by the soluble form of the receptor. The secretion in response to ATP was also inhibited by treatment with the endocytosis inhibitor dynasore, and by disruption of the acidic gradient of the endocytic compartment using different methods (chloroquine, NH4Cl or monensin). IL-6 secretion was also impaired by NED-19, a specific inhibitor of the two pore channels receptor mediating Ca 2+ release from the endolysosomal compartment. IL-6 and ATP increased IL-6 mRNA levels, an effect blocked by tocilizumab. Altogether, our results demonstrate that ATP-secreted IL-6 activates a positive loop based on IL-6 receptors, endocytosis, two pore channels and IL-6 transcription. Given the importance of muscle IL-6 as a systemic regulator and as an inflammatory mediator, our study can help to understand muscle pathophysiology., Competing Interests: Declaration of competing interest Authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Redox balance is essential for sustaining normal physiological metabolic activities of life. In this study, we present a photocatalytic system to perturb the balance of NADH/NAD + in oxygen-free conditions, achieving photocatalytic therapy to cure anaerobic bacterial infected periodontitis. Under light irradiation, the catalyst TBSMSPy + can bind bacterial DNA and initiate the generation of radical species through a multi-step electron transfer process. It catalyzes the conversion from NADH to NAD + (the turnover frequency up to 60.7 min -1 ), inhibits ATP synthesis, disrupts the energy supply required for DNA replication, and successfully accomplishes photocatalytic sterilization in an oxygen-free environment. The catalyst participates in the redox reaction, interfering with the balance of NADH/NAD + contents under irradiation, so we termed this action as photoinduced redox imbalance. Additionally, animal experiments in male rats also validate that the TBSMSPy + could effectively catalyze the NADH oxidation, suppress metabolism and stimulate osteogenesis. Our research substantiates the concept of photoinduced redox imbalance and the application of photocatalytic therapy, further advocating the development of such catalyst based on photoinduced redox imbalance strategy for oxygen-free phototherapy., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Although the αC-β4 loop is a stable feature of all protein kinases, the importance of this motif as a conserved element of secondary structure, as well as its links to the hydrophobic architecture of the kinase core, has been underappreciated. We first review the motif and then describe how it is linked to the hydrophobic spine architecture of the kinase core, which we first discovered using a computational tool, local spatial Pattern (LSP) alignment. Based on NMR predictions that a mutation in this motif abolishes the synergistic high-affinity binding of ATP and a pseudo substrate inhibitor, we used LSP to interrogate the F100A mutant. This comparison highlights the importance of the αC-β4 loop and key residues at the interface between the N- and C-lobes. In addition, we delved more deeply into the structure of the apo C-subunit, which lacks ATP. While apo C-subunit showed no significant changes in backbone dynamics of the αC-β4 loop, we found significant differences in the side chain dynamics of K105. The LSP analysis suggests disruption of communication between the N- and C-lobes in the F100A mutant, which would be consistent with the structural changes predicted by the NMR spectroscopy., Competing Interests: JW, NJ, SH, CO, GV, AK, ST No competing interests declared, (© 2023, Wu, Jonniya et al.)

PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and biolayer interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites., (© 2024 The Author(s).)

Human Schlafen 11 (SLFN11) is sensitizing cells to DNA damaging agents by irreversibly blocking stalled replication forks, making it a potential predictive biomarker in chemotherapy. Furthermore, SLFN11 acts as a pattern recognition receptor for single-stranded DNA (ssDNA) and functions as an antiviral restriction factor, targeting translation in a codon-usage-dependent manner through its endoribonuclease activity. However, the regulation of the various SLFN11 functions and enzymatic activities remains enigmatic. Here, we present cryo-electron microscopy (cryo-EM) structures of SLFN11 bound to tRNA-Leu and tRNA-Met that give insights into tRNA binding and cleavage, as well as its regulation by phosphorylation at S219 and T230. SLFN11 phosphomimetic mutant S753D adopts a monomeric conformation, shows ATP binding, but loses its ability to bind ssDNA and shows reduced ribonuclease activity. Thus, the phosphorylation site S753 serves as a conformational switch, regulating SLFN11 dimerization, as well as ATP and ssDNA binding, while S219 and T230 regulate tRNA recognition and nuclease activity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Miki Hara-Yokoyama reports financial support was provided by LiberoThera Co., Ltd. Shigeyuki Yokoyama reports a relationship with LiberoThera Co., Ltd that includes: funding grants. Kazue Terasawa reports a relationship with LiberoThera Co., Ltd that includes: employment. There is no patents to disclose relevant to this work. has patent NA pending to NA. There is no additional relationships or activities to declare. 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 Authors. Published by Elsevier Inc. All rights reserved.)

To understand why Chlamydia trachomatis (Ct) (L2/434/Bu) favors hypoxia, we examined the dynamics of infected cells using a glycolysis-related PCR array and metabolomic analysis, along with the perturbation of nucleotide synthesis. Our findings revealed that, compared to normoxia, hypoxia with infection significantly and selectively upregulates the expression of genes related to glycolysis, glycogen degradation, and the pentose phosphate pathway. Furthermore, hypoxia induced a significant decrease in metabolite levels, particularly methionine-related metabolites, independent of infection, indicating efficient metabolism under hypoxia. Additionally, the perturbation of nucleotide synthesis with adenosine derivatives impaired Ct growth. Collectively, our results suggest that Ct favors a hypoxic environment with efficient metabolism, in which Ct readily activates glycolysis responsible for stable nucleotide synthesis as well as ATP supply., Competing Interests: Declaration of competing interest We have no conflicts of interest in association with the present study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is a highly aggressive brain tumor with poor prognosis and high recurrence rates. The complex immune microenvironment of GBM is highly infiltrated by tumor-associated microglia and macrophages (TAM). TAMs are known to be heterogeneous in their functional and metabolic states and can transmit either protumoral or antitumoral signals to glioma cells. Here, we performed bulk RNA sequencing and single-cell RNA sequencing on samples from patients with GBM, which revealed increased ATP synthase expression and oxidative phosphorylation activity in TAMs located in the tumor core relative to the tumor periphery. Both in vitro and in vivo models displayed similar trends of augmented TAM mitochondrial activity, along with elevated mitochondrial fission, glucose uptake, mitochondrial membrane potential, and extracellular ATP (eATP) production by TAMs in the presence of GBM cells. Tumor-secreted factors, including GM-CSF, induced the increase in TAM eATP production. Elevated eATP in the GBM microenvironment promoted glioma growth and invasion by activating the P2X purinoceptor 7 (P2X7R) on glioma cells. Inhibition of the eATP-P2X7R axis attenuated tumor cell viability in vitro and reduced tumor size and prolonged survival in glioma-bearing mouse models. Overall, this study revealed elevated TAM-derived eATP in GBM and provided the basis for targeting the eATP-P2X7R signaling axis as a therapeutic strategy in GBM. Significance: Glioblastoma-mediated metabolic reprogramming in tumor-associated microglia increases ATP secretion that supports cancer cell proliferation and invasion by activating P2X7R, which can be inhibited to attenuate tumor growth., (©2024 American Association for Cancer Research.)

Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration., (© 2024 Masin et al.)

Piezo ion channels play a role in bladder sensation, but the sensory afferent subtypes that utilise Piezo channels have not been fully explored. We made single-unit extracellular recordings from mucosal-projecting bladder afferents in guinea pigs with protamine/zymosan-induced cystitis. The Piezo1 agonist, Yoda1, significantly potentiated mechanosensitivity, while its antagonist, Dooku1, abolished this potentiation. The P2 purinoceptor antagonist, PPADS abolished α,β-methylene ATP-induced excitation of mucosal afferents without affecting their mechanical activation or potentiation of mechanosensitivity by Yoda1. The findings suggest Piezo1, but not ATP, is required for mechanotransduction in bladder mucosal afferents in cystitis., Competing Interests: Declaration of competing interest The authors of “Piezo1, but not ATP, is involved in mechanotransduction by bladder mucosal afferents in cystitis” declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Current study was aimed to assess the β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) on delivery of cholesterol, and substitution of fructose and glycerol with trehalose on the ram semen cryosurvival. Samples were collected, diluted with Tris-citric acid-LDL extender, pooled, and used. In experiment I, βCD and MβCD carriers were used and compared to deliver the cholesterol (at 0, 0.5, 1, 1.5, 2, and 4 mg/mL). In the experiment II, trehalose (0, 7, 14, 21, and 28 mM) was substituted with fructose (28, 21, 14, 7, 0 mM, respectively). In the experiment III, the best cholesterol/carrier dose groups from the first experiment, were selected to be evaluated with the fructose/trehalose (14/14 mM) combination compared to fructose (28 mM) alone. The concentration of glycerol in the above-mentioned experiments was set at 4.5%. In the experiment IV, the effect of lowering glycerol (4% vs. 4.5%) was assessed using selected cholesterol/carrier groups. Kinematics, chromatin integrity, ATP contents, malondialdehyde amounts and viability were evaluated. Cholesterol (especially at 1.5 and 2 mg/mL) improved the kinematics and ATP levels using both carriers. The optimised amounts of trehalose (14 mM)/fructose(14 mM) reduced peroxidation and DNA fragmentation levels. Co-administration of optimised levels of cholesterol with trehalose/fructose did not show extra beneficial effects compared to each of them. Trehalose could not protect the spermatozoa at lower amounts of glycerol (4% vs. 4.5%). In conclusion, either the optimised levels of cholesterol (using βCD or MβCD carriers) or substitution of half of the fructose with the trehalose alone could lead to improvement in quality of frozen/thawed ram semen., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

The intracellular environment of skeletal muscle can develop pronounced hyperthermia and acidosis during strenuous exercise, and these alterations in the typical intracellular conditions have been shown to alter mitochondrial respiration. However, the impact of these conditions on ATP synthesis is poorly understood. We used Thoroughbred racehorses to test the hypothesis that both hyperthermia and acidosis decrease the rate of ATP synthesis, but that athletic conditioning mitigates this loss of phosphorylation capacity. Isolated mitochondria were harvested from skeletal muscle before and after a 9-wk racetrack conditioning program that increased whole body aerobic capacity by 19%, and oxidative phosphorylation capacity was tested ex vivo under normothermic and hyperthermic conditions, as well as normal pH and acidic pH created by the addition of lactic acid. In unfit horses, hyperthermia caused a 30-55% decrease in the rate of ATP synthesis and loss of phosphorylation efficiency (P/O ratio decreased from 4.2 to 1.7 during maximal oxidative phosphorylation). Aerobic conditioning resulted in increased phosphorylation efficiency under hyperthermic conditions. Lactic acidosis had a small negative effect on ATP synthesis in unfit horses, but aerobic conditioning increased the sensitivity of isolated mitochondria to the deleterious effects of lactic acidosis. These data support the prominent role of hyperthermia in skeletal muscle fatigue during exercise, particularly in unfit subjects. However, acidosis may be a more important cause of failure of ATP synthesis in fit subjects. NEW & NOTEWORTHY This study reports the use of a novel method for the measurement of ATP synthesis and concurrent mitochondrial respiration. Although acidosis is often considered a leading cause of exercise fatigue, this study shows that tissue hyperthermia, which often occurs concurrently with acidosis, has a larger role in decreasing the maximal rate of skeletal muscle ATP synthesis.

TND1128, a 5-deazaflavin derivative, is a drug with self-redox ability. We examined the effect of TND1128 on the level of mitochondrial membrane potential (ΔΨ m ), which is the most critical motive power for the biosynthesis of ATP. We prepared brain slices from mice pretreated with TND1128 (0.1-10 mg/kg, intraperitoneally) and detected ΔΨ m level with JC-1, a fluorescence ΔΨ m indicator. We further examined the depolarization of ΔΨ m under 5-min exposure to 25 mM KCl-ACSF (25K-ACSF), which activated neuronal voltage-dependent Ca 2+ channels. We evaluated the effect of TND1128 by using the inverse number of the ΔΨ m value as the ATP synthesis index (ASI). The level of ΔΨ m increased significantly by 24-h pretreatment with TND1128 (10 mg/kg), and significantly higher depolarization of the ΔΨ m was observed with 25K-ACSF exposure than in non-treated control. We found a significant decrease in 25K-ACSF induced [Ca 2+ ] c and [Ca 2+ ] m levels in the TND1128-pretreated preparations. We confirmed the dose and time-dependent facilitatory effects of TND1128 on the ASI. This study suggested that TND1128 could be incorporated into the TCA cycle and electron transfer chains to facilitate the polarization of ΔΨ m and activate on-demand ATP synthesis. TND1128 might rescue neurons in various brain diseases caused by energy defects. (198)., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Visceral leishmaniasis is caused by Leishmania donovani which affects the poorer sections of society, and despite the global spread, effective treatment is unavailable. The current study investigates the potential of leishmanial histidyl-tRNA synthetase (LdHisRS) as a drug target. LdHisRS delineated more closeness to other protozoan parasites than its mammalian counterparts and contained relevant differences in the active site residues. The important ATP-binding residues were mutated to alanine and all the proteins, including human HisRS, were purified to homogeneity. LdHisRS exhibited a dimeric state in solution and showed maximal amino acid activation activity in physiological conditions. It also demonstrated a greater affinity for substrate over cofactor, while magnesium and potassium enhanced its activity better than other tested metal ions. Comp-7m, a benzothiazolo-coumarin derivative, proved to be specific inhibitor of LdHisRS with competitive mode of inhibition for ATP whereas it displayed lower binding affinity towards mutants. LdHisRS majorly contained α-helices and most of the aromatic residues were present in its hydrophobic core. Additionally, Comp-7m superimposed on ATP adenine ring during docking analysis and LdHisRS-ligand complexes had comparable stability as well as rigidity in molecular dynamics simulation. We thus provide structural and functional insights of LdHisRS which can be useful for devising antileishmanials., 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.)

The fungicides strobilurins and succinate dehydrogenase inhibitors (SDHIs) are blockers of the electron transport chain (ETC) in fungi. Here, we show that the exposure for 24 h to kresoxym-methyl, a fungicide from the class of strobilurins, alters the mitochondrial respiration in human HepG2 hepatocytes. In addition, we demonstrate an increase in production of mitochondrial superoxide radical anion, a reduction in ATP level, a decrease in the ratio reduced/oxidized glutathione and a decrease in cell viability (assessed by the LDH assay, Presto Blue assay, and Crystal Violet assay). As kresoxym-methyl is associated to boscalid (SDHI) in commercial formulations, we analyzed a potential exacerbation of the induced mitochondrial dysfunction for this combination. For the highest dose at which kresoxym-methyl (5 µM) and boscalid (0.5 µM) did not induce changes in mitochondrial function when used separately, in contrast, when both fungicides were used in combination at the same concentration, we observed a significant alteration of the mitochondrial function of hepatocytes: there was a decrease in oxygen consumption rate, in the ATP level. In addition, the level of mitochondrial superoxide radical anion was increased leading to a decrease in the ratio reduced/oxidized glutathione, and an increase in viability.

Pyroptosis plays an important role in maintenance of intestinal homeostasis, the abnormal activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome can promote the event and development of ulcerative colitis (UC). Its protective effects such as inhibiting pyroptosis in various inflammation-related diseases have been demonstrated, but whether resveratrol (RES) can also alleviate the progression of the disease by inhibiting pyroptosis in UC and the mechanism have rarely been studied. In this study, lipopolysaccharide (LPS) combined with adenosine triphosphate (ATP) was used to induce HT29 human colon cancer cells to construct an intestinal epithelial cell pyroptosis and inflammation model in vitro to investigate the anti-inflammatory effect of RES, reveal the regulatory mechanism of RES on pyroptosis, and provide a new theoretical basis for the treatment of UC. In vitro experiences, HT29 cells were dividing into control group, LPS/ATP group, RES low-dose group, RES high-dose group, NF-κB inhibitor pyrrolidine dithiocarbamate group (PDTC group), and LPS/ATP+PDTC group. The mRNA expressions of pyroptosis-related indicators such as NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-1(CASP1), IL-18, IL-1β, and inflammatory factors such as TNF-α and IL-6 were detected by qRT-PCR. The protein expressions of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β, NF-κB-p65 in the nucleus, and IκBα and p-IκBα in the cytoplasm were detected by Western blot. Immunofluorescence saw the distribution and expression of NLRP3, ASC and NF-κB-p65 protein in each group. The morphology and degree of pyroptosis in each group were observed by transmission electron microscope. The results showed that compared with the control group, the pyroptosis-related proteins including NLRP3, ASC, CASP1, IL-18, IL-1β, and inflammatory factors including TNF-α and IL-6 in the LPS/ATP group were increased, and LPS/ATP activated the activity of NF-κB signaling pathway. Compared with the LPS/ATP group, RES downregulated the expression of pyroptosis-related proteins and inflammatory factors in HT29 cells, and inhibited the activation of the NF-κB signaling pathway in HT29 cells pyroptosis. RES down-regulates the pyroptosis of HT29 cells induced by LPS/ATP and the expression of pyroptosis-related indicators NLRP3, ASC, CASP1, IL-18, IL-1β and inflammatory factors TNF-α and IL-6 in the inflammatory response and inhibits the occurrence of pyroptosis. The mechanism is related to the inhibition of NF-κB pathway activity.

Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current flow in response to an applied voltage. Though, these methods are limited by low throughput, as they assess mitochondrial activity at a single time point under a specific treatment condition. This study explores, for the first time, the application of the Agilent Seahorse XFp Technology to evaluate metabolism in stallion spermatozoa. This method enables real-time measurement of cellular metabolism across multiple samples or experimental conditions simultaneously. Ejaculates from eight different stallions were collected, and pools were prepared from three of them. Sperm viability and mitochondrial activity were evaluated by fluorescence microscopy, sperm motility by a computer-assisted sperm analysis system, and sperm metabolism was analysed via the Seahorse XFp analyser. Results confirmed a preference for OXPHOS over glycolysis in ATP production in stallion sperm, with mitochondria contributing significantly to total ATP generation. The Seahorse XFp Technology proved effective in evaluating equine sperm bioenergetics, offering insights into metabolic pathways critical for sperm function. In conclusion, this technology grants a new method for high-throughput analysis of sperm metabolism and quality, which could be applied to future reproductive studies in male equine fertility., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: The objective of this study was to investigate the protective mechanism of Ling-Gui-Zhu-Gan decoction (LGZGD) against LPS-ATP-induced pyroptosis in H9c2 cells., Methods: LPS and ATP were used to induce pyroptosis in the H9c2 cell, and the cells were divided into the control, model and LGZGD groups. LDH level was detected using a colorimetric assay. ELISA was used to detect the expressions of IL-1β. Flow cytometry was utilized to observe apoptosis, while Hoechst/PI staining was used to detect pyroptosis. Immunofluorescence was employed to observe the expression levels of NLRP3 in cardiomyocytes, and RT-PCR was used to detect NLRP3, Caspase-1, GSDMD, and ASC mRNA expression. The cells were separated into seven groups: control, model, LGZGD, MCC950, LGZGD+MCC950, Nigericin and LGZGD+Nigericin. The mRNA and protein expressions were determined by RT-PCR and Western blot., Results: LPS (10 μg/mL) for 12 h and ATP (8 mM) for 2 h were used as modeling condition. LGZGD demonstrated a significant reduction in LDH, and IL-1β levels (P<0.05, P<0.01). LGZGD dramatically reduced apoptosis rate, inhibited pyroptosis, decreased the fluorescence expressions of NLRP3, and reduced the mRNA expressions of NLRP3, ASC, Caspase-1, and GSDMD (P<0.01). Further mechanism studies showed that NLRP3, ASC, Caspase-1, and GSDMD decreased significantly when combined with NLRP3 inhibitor MCC950. Furthermore, LGZGD was able to effectively reverse the upregulation of protein and gene expression of Nigericin group (P<0.01)., Conclusion: LGZGD inhibits LPS-ATP-induced pyroptosis in H9c2 cell via the NLRP3/Caspase-1 signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Mammary epithelial cells (MECs) of humans (h) and Göttingen Minipigs (mp) were analyzed to compare their ability to perform ATP production by oxidative phosphorylation and glycolysis. The ATP production under basal and stressor situations highlights the same metabolic potential of both primary cell lines. However, quantitively the ATP production rate of hMECs was higher than mpMECs. Conversely, oxidative cell respiration in mpMECs exploits a maximum respiratory capacity to support pathophysiological circumstances or stress conditions that could require an excessive effort of cell metabolism. Since mpMECs primarily utilize an oxidative metabolism similar to hMECs, the metabolic characterization conducted allows us to confirm that mpMECs represent a potential alternative cellular model in the translational medicine approach., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Salvatore Nesci reports financial support was provided by University of Bologna. 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.)

Some DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Candida albicans is the most common pathogen in systemic fungal diseases, exhibits a complex pathogenic mechanism, and is increasingly becoming drug tolerant. Therefore, it is particularly important to study the genes associated with virulence and resistance of C. albicans . Here, we identified a gene ( orf19.1588 ) that encodes a conserved mitochondrial protein known as CaSDH8 , upon deletion of CaSdh8 , the deleted strain ( Casdh8Δ/Δ ) experienced impaired growth, hyphal development, and virulence. Casdh8Δ/Δ displayed a reduced capacity to utilize alternative carbon sources, along with detrimental alterations in reactive oxygen species (ROS), mitochondrial membrane potential (MMP) depolarization, and adenosine triphosphate (ATP) levels. Interestingly, Casdh8Δ/Δ demonstrated resistance to azole drugs, and under the influence of fluconazole, the cell membrane permeability and mitochondrial function of Casdh8Δ/Δ were less compromised than those of the wild type, indicating a reduction in the detrimental effects of fluconazole on Casdh8Δ/Δ . These findings highlight the significance of CaSDH8 as a crucial gene for the maintenance of cellular homoeostasis. Our study is the first to document the effects of the CaSDH8 gene on the virulence and azole resistance of C. albicans at both the molecular and animal levels, providing new clues and directions for the antifungal infection and the discovery of antifungal drug targets.

Protein kinase inhibitors designed to compete with ATP as a primary mode of action turn out to have considerable effects that go beyond their interference of nucleotide binding. New research shows how kinase activation and sometimes noncatalytic functions of protein kinases can be controlled by allosteric properties of kinase inhibitors, communicating perturbations from the active site to distal regulatory regions., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Zymosan is a β-glucan-rich component derived from the cell walls of Saccharomyces cerevisiae extensively used in research for its potent immunomodulatory properties. It can prompt inflammatory responses such as peritonitis and arthritis, and is particularly used to study the immune response to fungal particles. Although the zymosan induced-release of the proinflammatory cytokine IL-1β by macrophages is an essential mechanism for combating fungal infection and inducing inflammation, the exact processes leading to its release remain not well understood. In this study, we uncover the intracellular mechanisms involved in zymosan induced-release of active IL-1β by peritoneal macrophages. Zymosan initiates pro-IL-1β formation through TLR2/MyD88 activation; however, Dectin-1 activation only amplify the conversion of pro-IL-1β into its active form. The conversion of inactive to active IL-1β upon zymosan stimulation depends on the NLRP3, ASC, and caspase-1 driven by the decrease in intracellular potassium ions. Notably, zymosan-induced activation of caspase-1 does not require phagocytosis. Instead, zymosan induces a rapid drop in the intracellular ATP concentration, which occurs concomitant with caspase-1 and IL-1β activation. Accordingly, disruption of glycolytic flux during zymosan stimulation promotes an additional reduction of intracellular ATP and concurrently amplifies the activation of caspase-1 and IL-1β. These results reveal that fungal recognition by macrophages results in a metabolic dysfunction, leading to a decrease of intracellular ATP associated with inflammasome activation., Competing Interests: Declaration of competing interest Authors have no competing interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

With age, metabolic perturbations accumulate to elevate our obesity burden. While age-onset obesity is mostly driven by a sedentary lifestyle and high calorie intake, genetic and epigenetic factors also play a role. Among these, members of the large histone deacetylase (HDAC) family are of particular importance as key metabolic determinants for healthy ageing, or metabolic dysfunction. Here, we aimed to interrogate the role of class 2 family member HDAC5 in controlling systemic metabolism and age-related obesity under non-obesogenic conditions. Starting at 6 months of age, we observed adult-onset obesity in chow-fed male global HDAC5-KO mice, that was accompanied by marked reductions in adrenergic-stimulated ATP-consuming futile cycles, including BAT activity and UCP1 levels, WAT-lipolysis, skeletal muscle, WAT and liver futile creatine and calcium cycles, and ultimately energy expenditure. Female mice did not differ between genotypes. The lower peripheral sympathetic nervous system (SNS) activity in mature male KO mice was linked to higher dopaminergic neuronal activity within the dorsomedial arcuate nucleus (dmARC) and elevated hypothalamic dopamine levels. Mechanistically, we reveal that hypothalamic HDAC5 acts as co-repressor of STAT5b over the control of Tyrosine hydroxylase (TH) gene transactivation, which ultimately orchestrates the activity of dmARH dopaminergic neurons and energy metabolism in male mice under non-obesogenic conditions., Competing Interests: Declaration of competing interest The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for Molecular Metabolism and was not involved in the editorial review or the decision to publish this article. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PTP received speaker honoraria by Novo Nordisk. As a scientist, MHT participated in a scientific advisory board meeting of ERX Pharmaceuticals, Inc., Cambridge, MA, in 2019. He was a member of the Research Cluster Advisory Panel (ReCAP) of the Novo Nordisk Foundation between 2017 and 2019. He received funding for his research projects by Novo Nordisk (2016–2020) and Sanofi-Aventis (2012–2019). He consulted twice for Böhringer Ingelheim Pharma GmbH & Co. KG (2020 & 2021) and delivered a scientific lecture for Sanofi-Aventis Deutschland GmbH (2020) and for AstraZeneca GmbH (2024). As CEO and CSO of Helmholtz Munich, he is co-responsible for countless collaborations of the employees with a multitude of companies and institutions, worldwide. In this capacity, he discusses potential projects with and has signed/signs contracts for the centers institute(s) related to research collaborations worldwide, including but not limited to pharmaceutical corporations like Boehringer Ingelheim, Novo Nordisk, Roche Diagnostics, Arbormed, Eli Lilly, SCG Cell Therapy and others. As the CEO of Helmholtz Munich, he was/is further overall responsible for commercial technology transfer activities. MHT confirms that to the best of his knowledge none of the above funding sources or collaborations were involved in or had an influence on the preparation of this manuscript. All other authors declare that they have no conflict of interest related to this study., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The contamination by the toxin citrinin (CIT), produced by fungi, has been reported in agricultural foods and is known to be nephrotoxic to humans. In this study, we found that CIT could be effectively degraded by the oleaginous yeast Saitozyma podzolica zwy-2-3. Four genes encoding glycosyltransferases (GTs) in S. podzolica zwy-2-3 (SPGTs) were identified by evolutionary and structural analyses. The overexpression of SPGTs enhanced CIT degradation to 0.56 mg/L/h in S. podzolica zwy-2-3 by increasing ATP and glutathione (GSH) contents to oxidize CIT and scavenge reactive oxygen species (ROS). Besides, SPGTs promoted lipid synthesis by 9.3 % of S. podzolica zwy-2-3 under CIT stress. These results suggest that SPGTs in oleaginous yeast play a pivotal role in enhancing CIT degradation and lipid accumulation. These findings provide a valuable basis for the application of GTs in oleaginous yeast to alleviate CIT contamination in agricultural production, which may contribute to food safety., 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 endoplasmic reticulum-mitochondrial encounter structure (ERMES) complex is known to play crucial roles in various cellular processes. However, its functional significance in filamentous fungi, particularly its impact on deoxynivalenol (DON) biosynthesis in Fusarium graminearum, remains inadequately understood. In this study, we aimed to investigate the regulatory function of the ERMES complex in F. graminearum. Our findings indicate significant changes in mitochondrial morphology of ERMES mutants, accompanied by decreased ATP content and ergosterol production. Notably, the toxisome formation in the ERMES mutant ΔFgMDM10 was defective, resulting in a substantial reduction in DON biosynthesis. This suggests a pivotal role of ERMES in toxisome formation, as evidenced by the pronounced inhibition of toxisome formation when ERMES was disrupted by boscalid. Furthermore, ERMES deficiencies were shown to diminish the virulence of F. graminearum towards host plants significantly. In conclusion, our results suggest ERMES is an important regulator of mitochondrial morphology, DON biosynthesis, and toxisome formation in F. graminearum., Competing Interests: Declaration of Competing Interest All the authors have no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The severity, frequency, and duration of extreme events, in the context of global warming, have placed many marine ecosystems at high risk. Therefore, the application of methods that can mediate the impacts of global warming on marine organisms seems to be an emerging necessity in the near term. In this context, enhancing the thermal resilience of marine organisms may be crucial for their sustainability. It has been shown that the repeated time-limited exposure of an organism to an environmental stimulus modifies its response mode, thus enhancing resilience and allowing adaptation of the physiological and developmental phenotype to environmental stress. In the present study, we investigated the "stress memory" effect caused by heat hardening on Mytilus galloprovincialis cellular pathways to identify the underlying biochemical mechanisms that enhance mussel thermal tolerance. Heat hardening resulted in increased ETS activity and ATP production and increased autophagic performance at all elevated temperatures (24 °C, 26 °C, and 28 °C). Furthermore, at these increased temperatures, apoptosis and inflammation remain at significantly lower levels in pregnant individuals than in nonhardened individuals. Autophagy, as a negative regulator of apoptosis, may lead to decreased damage to surrounding cells, which in turn alleviates inflammatory effects. In conclusion, the exposure of mussels to heat hardening seems to provide a physiological response that enhances heat tolerance and increases cell survival through increased energy production and reduced cell death and inflammatory responses. The latter can be utilized for the management and conservation of aquatic species of economic value or endangered status., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Risperidone (RIS) is a widely used antipsychotic drug with reported alteration in immune response. The current study investigated mitochondrial disruption as the underlying mechanism of RIS-induced immunotoxicity in isolated human peripheral blood monocytes (hPBM). RIS was cytotoxic to hPBM in exposure duration and concentration-dependent patterns. Functionally, RIS was shown to increase the release of IL-6, TNF-α, and IL-8 with a decrease in test particle phagocytosis in concertation and exposure time-based patterns. It was found that RIS decreased ATP production in isolated monocytes' mitochondria, with an estimated EC50 of around 70 μM after 24 h with parallel inhibition of mitochondrial complexes I and III activities and decreased mitochondrial membrane potential and oxygen consumption rates with increased lactate production from by the treated cells in comparison to controls. Structurally, RIS in 100 μM concentration significantly increased the mitochondrial membrane fluidity with significant increase in increased unsaturated/saturated fatty acids ratios of the mitochondrial membranes of the treated cells. Interestingly, water-soluble CoQ10 formulation significantly decreased the cytotoxic effect of RIS and improved the phagocytic activity of RIS-treated cells. To conclude, the current data suggests mitochondrial disruption as the underlying mechanism of RIS-induced immunotoxicity with shown protective effect of water-soluble CoQ10 formulation., 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 complex regional pain syndrome type 1 (CRPS-1) is one of the most discussed painful syndromes due to the variability and severity of its symptoms. CRPS-1 generally occurs after a trauma, a fracture or a sprain followed by an immobilization. Classical diagnostic criteria are not always clear; hence, the diagnosis is difficult. The definition of CRPS itself defines and considers the pain as key symptom neglecting the bone damage. Early CRPS involves the activation of the innate cutaneous immune system with altered sensory and sympathetic signaling, activation and proliferation of keratinocytes and mast cells in addition to the release of inflammatory mediators and pain. The role of the immune system and the response to the disease is becoming clearer as the microglia is activated as a result of injury and can induce a central sensitization while astrocytes can maintain the process. Adenosine triphosphate (ATP) exerts a fundamental role in the activation of innate cutaneous immune system, in the proliferation of keratinocytes and mast cells, in the release of several proinflammatory cytokines and in the microglia activation. It is essential to intervene on this pathology as soon as possible with drugs, as clodronate, able to reduce bone marrow edema and pain through the inhibition of the primary inflammatory process and the immune reaction, limiting the activation of macrophages and the release of cytokines activating nuclear growth factor (NGF). In this review the role of ATP, bisphosphonates and rehabilitation are discussed.

Monitoring and quantifying ATP levels in vivo is essential to understanding its role as a signaling molecule in tumor progression and therapy. Nevertheless, the real-time monitoring and quantitative assessment of lysosomal ATP remains challenging due to the lack of accurate tools in deep tissues. In this study, based on the crosslinking enhanced emission (CEE) effect, we successfully synthesized red carbon dots (R-CDs) with dual emission properties for efficient quantification of intracellular ATP. The R-CDs emit in the near-infrared range and target lysosomes with rapid detection capabilities, rendering them exceptionally well-suited for directly observing and analyzing the dynamics of lysosomal ATP through live cell imaging techniques. Importantly, R-CDs have proven their efficacy in real-time monitoring of drug stimulus-induced fluctuations in endogenous lysosomal ATP concentration and have also been employed for quantifying and distinguishing lysosomal ATP levels among normal and cancer cell lines. These noteworthy findings emphasize the versatility of the R-CD as a valuable imaging tool for elucidating the functional role of lysosomal ATP in drug screening and cancer diagnostics and hold the promise of becoming a reference tool for deepening our understanding of drug mechanisms of action., 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.)

During the asymmetric loop extrusion of DNA by a condensin complex, one domain of the complex stably anchors to the DNA molecule, and another domain reels in the DNA strand into a loop. The DNA strand in the loop is fully relaxed, or there is no tension in the loop. Just outside of the loop, there is a tension that resists the extrusion of DNA. To maintain the extrusion of the DNA loop, the condensin complex must have a domain capable of generating a force to overcome the tension outside of the loop. This study proposes that the groove-shaped HEAT repeat domain Ycg1 plays the role of a molecular motor. A DNA molecule may bind to the groove electrostatically, and the weak binding force facilitates the random thermal motion of DNA molecules. A mechanical model that random collisions between DNA and the nonparallel inner surfaces of the groove may generate a directional force which is required for the loop extrusion to sustain. The hinge domain binds to the DNA molecule and acts as an anchor during asymmetric DNA loop extrusion. When the effects of ATP hydrolysis and the viscous drag of the fluid environment are considered, the motor-anchor model for the condensin complex and the mechanical model might explain the asymmetric loop extrusion, the formation of steps, the step size distribution in the loop extrusion, the tension-dependent extrusion speed, the interaction between coexisting loops on the DNA strand, and untying the knots during extrusion. This model can also explain the observed formation of the Z-loop., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

One of the leading causes of cancer-related deaths worldwide is colorectal cancer (CRC). Extracellular ATP (e-ATP) and purinergic receptors (P2R) play a central role in CRC proliferation and progression. Human antigen R (HuR) is becoming more and more understood to be essential for the expression of genes linked to cancer. The current study demonstrates that ATP can mediate CRC (Caco-2 cells) progression via induction of HuR nucleocytoplasmic shuttling and subsequent expression of cancer-related genes, a consequence mostly mediated via the P2R receptor. It was also noted that suppression of HuR activity by using dihydrotanshinone I (DHTS) prevents cancer-related gene expression and subsequent CRC (Caco-2 cells) progression induced by ATP. The expression of cyclin A2/cyclin-dependent kinase 2 (CDK2), Bcl-2, ProT-α, hypoxia-inducible factor1-α (HIF1-α), vascular endothelial growth factor A (VEGF-A), transforming growth factor-β (TGF-β) and matrix metallopeptidase 9 (MMP-9) induced by ATP were highly reduced in the presence of either PPADS (non-selective P2R antagonist) or DHTS. In addition, e-ATP-induced Caco-2 cell proliferation as well as cell survival were highly reduced in the presence of either PPADS or DHTS or selective CDK-2 inhibitor (Roscovitine) or selective Bcl-2 inhibitor (ABT-263). Furthermore, it was found that MMP-9 is critical for Caco-2 cells migration induced by e-ATP as demonstrated by a clear reduction in cells migration in the presence of a selective MMP-9 inhibitor (Marimastat). Collectively, these data demonstrate that ATP through P2R activation can induce HuR nucleocytoplasmic shuttling that could be translated into an increase in cancer-related genes expression and subsequent, cell proliferation and progression., Competing Interests: Declarations Competing interests All of the authors declare that there are no conflicting interests., (© 2024. The Author(s).)

Context: Sepsis-induced acute lung injury (ALI) is a severe condition with limited effective therapeutics; nicotinamide mononucleotide (NMN) has been reported to exert anti-inflammatory activities., Objective: This study explores the potential mechanisms by which NMN ameliorates sepsis-induced ALI in vivo and in vitro ., Materials and Methods: Cultured MH-S cells and a murine model were used to evaluate the effect of NMN on sepsis-induced ALI. MH-S cells were stimulated with LPS (1 μg/mL) and NMN (500 μM) for 12 h grouping as control, LPS, and LPS + NMN. Cell viability, apoptotic status, and M1/2 macrophage-related markers were detected. The mice were pretreated intraperitoneally with NMN (500 mg/kg) and/or EX-527 (5 mg/kg) 1 h before LPS injection and randomized into 7 groups ( n  = 8): control, LPS, LPS + NMN, NMN, LPS + NMN + EX-527 (a SIRT1 inhibitor), LPS + EX-527, and EX-527. After 12 h, lung histopathology, W/D ratio, MPO activity, NAD + and ATP levels, M1/2 macrophage-related markers, and expression of the SIRT1/NF-κB pathway were detected., Results: In MH-S cells, NMN significantly decreased the apoptotic rate from 12.25% to 5.74%. In septic mice, NMN improved the typical pathologic findings in lungs and reduced W/D ratio and MPO activity, but increased NAD + and ATP levels. Additionally, NMN suppressed M1 but promoted M2 polarization, and upregulated the expression of SIRT1, with inhibition of NF-κB-p65 acetylation and phosphorylation. Furthermore, inhibition of SIRT1 reversed the effects of NMN-induced M2 macrophage polarization., Conclusions: NMN protects against sepsis-induced ALI by promoting M2 macrophage polarization via the SIRT1/NF-κB pathway, it might be an effective strategy for preventing or treating sepsis-induced ALI.

Introduction: Insulin secretion is a highly regulated process critical for maintaining glucose homeostasis. This abstract explores the intricate interplay between three essential pathways: The Triggering Pathway, The Metabolic Amplifying Pathway, and Cellular Transduction, in orchestrating glucose-dependent biphasic insulin secretion., Mechanism: During the triggering pathway, glucose metabolism in pancreatic beta-cells leads to ATP production, closing ATP-sensitive potassium channels and initiating insulin exocytosis. The metabolic amplifying pathway enhances insulin secretion via key metabolites like NADH and glutamate, enhancing calcium influx and insulin granule exocytosis. Additionally, the cellular transduction pathway involves G-protein coupled receptors and cyclic AMP, modulating insulin secretion., Result and Conclusion: These interconnected pathways ensure a dynamic insulin response to fluctuating glucose levels, with the initial rapid phase and the subsequent sustained phase. Understanding these pathways' complexities provides crucial insights into insulin dysregulation in diabetes and highlights potential therapeutic targets to restore glucose-dependent insulin secretion.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (k cat ). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site., (© 2024 The Authors. Cytoskeleton published by Wiley Periodicals LLC.)

To further reveal the inhibition mechanism of carbon dioxide (CO 2 ) on Shewanella putrefaciens (S. putrefaciens), influence on metabolic function was studied by biochemical and metabolomics analysis. Accordingly, reduction of intracellular pH (pH i ), depolarization of cell membrane and accumulation of reactive oxygen species (ROS) indicated that CO 2 changed the membrane permeability of S. putrefaciens. Besides, adenosine triphosphate (ATP), ATPase, nicotinamide adenine dinucleotide (NAD + /NADH) and ratios of NADH/NAD + were detected, indicating a role of CO 2 in repressing respiratory pathway and electron transport. According to metabolomics results, CO 2 induced differential expressions of metabolites, disordered respiratory chain and weakened energy metabolism of S. putrefaciens. Inhibition of respiratory rate-limiting enzymes also revealed that electron transfer of respiratory chain was blocked, cell respiration was weakened, and thus energy supply was insufficient under CO 2 stress. These results revealed that CO 2 caused disruption of metabolic function, which might be the main cause of growth inhibition for S. putrefaciens., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Ltd.)

There is growing interest in the P2X4 receptor as a therapeutic target for several cardiovascular, inflammatory and neurological conditions. Key to exploring the physiological and pathophysiological roles of P2X4 is access to selective compounds to probe function in cells, tissues and animal models. There has been a recent growth in selective antagonists for P2X4, though agonist selectivity is less well studied. As there are some known pharmacological differences between P2X receptors from different species, it is important to understand these differences when designing a pharmacological strategy to probe P2X4 function in human tissue and mouse models. Here, we provide a systematic comparison of agonist and antagonist pharmacology in 1321N1 cells expressing either human or mouse P2X4 orthologues. We identify a rank order of agonist potency of ATP > 2-MeSATP > αβmeATP = BzATP > CTP = γ-[(propargyl)-imido]-ATP for human P2X4 and ATP > 2-MeSATP = CTP > ATPγS = γ-[(propargyl)-imido]-ATP = BzATP for mouse. Human P2X4 is not activated by ATPγS but can be activated by αβmeATP. We identify a rank order of antagonist potency of BAY-1797 = PSB-12062 = BX-430 > 5-BDBD > TNP-ATP = PPADS for human P2X4 and BAY-1797 > PSB-12062 = PPADS > TNP-ATP for mouse. Mouse P2X4 is not antagonised by 5-BDBD or BX-430. The study reveals key pharmacological differences between human and mouse P2X4, highlighting caution when selecting tools for comparative studies between human and mouse and ascribing cellular responses of some commonly used agonists to P2X4., Competing Interests: Declarations Ethical approval Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The global surge in multidrug-resistant bacteria owing to antibiotic misuse and overuse poses considerable risks to human and animal health. With existing antibiotics losing their effectiveness and the protracted process of developing new antibiotics, urgent alternatives are imperative to curb disease spread. Notably, improving the bactericidal effect of antibiotics by using non-antibiotic substances has emerged as a viable strategy. Although reduced nicotinamide adenine dinucleotide (NADH) may play a crucial role in regulating bacterial resistance, studies examining how the change of metabolic profile and bacterial resistance following by exogenous administration are scarce. Therefore, this study aimed to elucidate the metabolic changes that occur in Edwardsiella tarda ( E. tarda ), which exhibits resistance to various antibiotics, following the exogenous addition of NADH using metabolomics. The effects of these alterations on the bactericidal activity of neomycin were investigated. NADH enhanced the effectiveness of aminoglycoside antibiotics against E. tarda ATCC15947, achieving bacterial eradication at low doses. Metabolomic analysis revealed that NADH reprogrammed the ATCC15947 metabolic profile by promoting purine metabolism and energy metabolism, yielding increased adenosine triphosphate (ATP) levels. Increased ATP levels played a crucial role in enhancing the bactericidal effects of neomycin. Moreover, exogenous NADH promoted the bactericidal efficacy of tetracyclines and chloramphenicols. NADH in combination with neomycin was effective against other clinically resistant bacteria, including Aeromonas hydrophila, Vibrio parahaemolyticus , methicillin-resistant Staphylococcus aureus , and Listeria monocytogenes . These results may facilitate the development of effective approaches for preventing and managing E. tarda -induced infections and multidrug resistance in aquaculture and clinical settings.

P2X7 is a member of the Ionotropic Purinergic Receptor (P2X) family. The P2X family of receptors is composed of seven (P2X1-7), ligand-gated, nonselective cation channels. Changes in P2X expression have been reported in multiple disease models. P2Xs have large complex extracellular domains that function as receptors for a variety of ligands, including endogenous and synthetic agonists and antagonists. ATP is the canonical agonist. ATP affinity ranges from nanomolar to micromolar for most P2XRs, but P2X7 has uniquely poor ATP affinity. In many physiological settings, it may be difficult to achieve the millimolar extracellular ATP concentrations needed for P2X7 channel activation; however, channel function is implicated in pain sensation, immune cell function, cardiovascular disease, cancer, and osteoporosis. Multiple high-resolution P2X7 structures have been solved in apo-, ATP-, and antagonist-bound states. P2X7 structural data reveal distinct allosteric and orthosteric antagonist-binding sites. Both allosteric and orthosteric P2X7 antagonists are well documented to inhibit ATP-evoked channel current. However, a growing body of evidence supports P2X7 activation by non-nucleotide agonists, including extracellular histone proteins and human cathelicidin-derived peptides (LL-37). Interestingly, P2X7 non-nucleotide agonism is not inhibited by allosteric antagonists, but is inhibited by orthosteric antagonists. Herein, we review P2X7 function with a focus on the efficacy of available pharmacology on P2X7 channel current activation by non-nucleotide agonists in effort to understand agonist/antagonist efficacy, and consider the impact of these data on the current understanding of P2X7 in physiology and disease given these limitations of P2X7-selective antagonists and incomplete knockout mouse models.