Endothelial calcium dynamics elicited by ATP release from red blood cells.

Red blood cells (RBCs) exhibit an interesting response to hydrodynamic flow, releasing adenosine triphosphate (ATP). Subsequently, these liberated ATP molecules initiate a crucial interaction with endothelial cells (ECs), thereby setting off a cascade involving the release of calcium ions (Ca 2 + ). Ca 2 + exerts control over a plethora of cellular functions, and acts as a mediator for dilation and contraction of blood vessel walls. This study focuses on the relationship between RBC dynamics and Ca 2 + dynamics, based on numerical simulations under Poiseuille flow within a linear two-dimensional channel. It is found that the concentration of ATP depends upon a variety of factors, including RBC density, channel width, and the vigor of the flow. The results of our investigation reveals several features. Firstly, the peak amplitude of Ca 2 + per EC escalates in direct proportion to the augmentation of RBC concentration. Secondly, increasing the flow strength induces a reduction in the time taken to reach the peak of Ca 2 + concentration, under the condition of a constant channel width. Additionally, when flow strength remains constant, an increase in channel width corresponds to an elevation in calcium peak amplitude, coupled with a decrease in peak time. This implies that Ca 2 + signals should transition from relatively unconstrained channels to more confined pathways within real vascular networks. This notion gains support from our examination of calcium propagation in a linear channel. In this scenario, the localized Ca 2 + release initiates a propagating wave that gradually encompasses the entire channel. Notably, our computed propagation speed agrees with observations.

Programmable melanoma-targeted radio-immunotherapy via fusogenic liposomes functionalized with multivariate-gated aptamer assemblies.

Radio-immunotherapy exploits the immunostimulatory features of ionizing radiation (IR) to enhance antitumor effects and offers emerging opportunities for treating invasive tumor indications such as melanoma. However, insufficient dose deposition and immunosuppressive microenvironment (TME) of solid tumors limit its efficacy. Here we report a programmable sequential therapeutic strategy based on multifunctional fusogenic liposomes (Lip@AUR-ACP-aptPD-L1) to overcome the intrinsic radio-immunotherapeutic resistance of solid tumors. Specifically, fusogenic liposomes are loaded with gold-containing Auranofin (AUR) and inserted with multivariate-gated aptamer assemblies (ACP) and PD-L1 aptamers in the lipid membrane, potentiating melanoma-targeted AUR delivery while transferring ACP onto cell surface through selective membrane fusion. AUR amplifies IR-induced immunogenic death of melanoma cells to release antigens and damage-associated molecular patterns such as adenosine triphosphate (ATP) for triggering adaptive antitumor immunity. AUR-sensitized radiotherapy also upregulates matrix metalloproteinase-2 (MMP-2) expression that combined with released ATP to activate ACP through an "and" logic operation-like process (AND-gate), thus triggering the in-situ release of engineered cytosine-phosphate-guanine aptamer-based immunoadjuvants (eCpG) for stimulating dendritic cell-mediated T cell priming. Furthermore, AUR inhibits tumor-intrinsic vascular endothelial growth factor signaling to suppress infiltration of immunosuppressive cells for fostering an anti-tumorigenic TME. This study offers an approach for solid tumor treatment in the clinics.

The physiological levels of epigallocatechin gallate (EGCG) enhance the Cd-induced oxidative stress and apoptosis in CHO-K1 cells.

Currently, the increasing pollution of the environment by heavy metals is observed, caused both by natural factors and those related to human activity. They pose a significant threat to human health and life. It is therefore important to find an effective way of protecting organisms from their adverse effects. One potential product showing a protective effect is green tea. It has been shown that EGCG, which is found in large amounts in green tea, has strong antioxidant properties and can therefore protect cells from the adverse effects of heavy metals. Therefore, the aim of the study was to investigate the effect of EGCG on cells exposed to Cd. In the study, CHO-K1 cells (Chinese hamster ovary cell line) were treated for 24 h with Cd (5 and 10 µM) and EGCG (0.5 and 1 µM) together or separately. Cell viability, ATP content, total ROS activity, mitochondrial membrane potential and apoptosis potential were determined. The results showed that, in tested concentrations, EGCG enhanced the negative effect of Cd. Further analyses are needed to determine the exact mechanism of action of EGCG due to the small number of publications on the subject and the differences in the results obtained in the research.

The toxic effects induced by benzethonium chloride on Daphnia carinata over two generations: Resistance and higher sublethal toxicity.

With the widespread utilization of the sanitizing product benzethonium chloride (BEC) throughout the coronavirus pandemic, concerns have emerged regarding its potential hazards. Nevertheless, the long-term and multigenerational toxic effects of BEC on aquatic organisms remains unexplored. This study investigates acute and chronic toxicity, oxidative stress, mitochondrial membrane potential, ATP concentrations, and gene expression using Daphnia carinata as the model organism. Meanwhile, hierarchical clustering analysis was utilized to investigate phenotypic effects among different treatment groups. The integrated biomarker response index version 2 (IBRv2) was employed to estimate the deviation in toxic effects over two generations. These results indicated that D. carinata in the second generation exhibited higher survival rate and lower levels of oxidative stress than the first generation. However, the higher sublethal effects were found in the second generation as follows, the weakened growth performance, mitochondrial membrane potential depolarization, reduced ATP concentrations, and down-regulated gene expression. The mitochondrial toxicity induced by BEC may account for the distinct toxic effects exhibited in two generations. The findings here can assist with the evaluation of potential risk for BEC on aquatic organisms, and provide new insight into the cross-generational toxicity mechanisms of pollutants in aquatic ecosystems.

Influence of extracellular ATP on mammalian sperm physiology.

In addition to its central role in cellular metabolism, adenosine 5'-triphosphate (ATP) is an important extracellular signalling molecule involved in various physiological processes. In reproduction, extracellular ATP participates in both autocrine and paracrine paths regulating gametogenesis, gamete maturation and fertilisation. This review focusses on how extracellular ATP modulates sperm physiology with emphasis on the mammalian acrosome reaction. The presence of extracellular ATP in the reproductive tract is primarily determined by the ion channels and transporters that influence its movement within the cells comprising the tract. The main targets of extracellular ATP in spermatozoa are its own transporters, particularly species-specific sperm purinergic receptors. We also discuss notable phenotypes from knock-out mouse models and human Mendelian inheritance related to ATP release mechanisms, along with immunological, proteomic, and functional observations regarding sperm purinergic receptors and their involvement in sperm signalling.

Single-molecule reconstruction of eukaryotic factor-dependent transcription termination.

Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism.

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

L-Theanine Prolongs the Lifespan by Activating Multiple Molecular Pathways in Ultraviolet C-Exposed Caenorhabditis elegans.

L-theanine, a unique non-protein amino acid, is an important bioactive component of green tea. Previous studies have shown that L-theanine has many potent health benefits, such as anti-anxiety effects, regulation of the immune response, relaxing neural tension, and reducing oxidative damage. However, little is known concerning whether L-theanine can improve the clearance of mitochondrial DNA (mtDNA) damage in organisms. Here, we reported that L-theanine treatment increased ATP production and improved mitochondrial morphology to extend the lifespan of UVC-exposed nematodes. Mechanistic investigations showed that L-theanine treatment enhanced the removal of mtDNA damage and extended lifespan by activating autophagy, mitophagy, mitochondrial dynamics, and mitochondrial unfolded protein response (UPRmt) in UVC-exposed nematodes. In addition, L-theanine treatment also upregulated the expression of genes related to mitochondrial energy metabolism in UVC-exposed nematodes. Our study provides a theoretical basis for the possibility that tea drinking may prevent mitochondrial-related diseases.

ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction.

Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.

CRISPR/Cas12a trans-cleavage mediated photoelectrochemical biosensor based on zeolitic imidazolate framework-67 for ATP determination.

An efficient PEC biosensor is proposed for ATP detection based on exciton energy transfer from CdTe quantum dots (CdTe QDs) to Au nanoparticles (AuNPs), integrating CRISPR/Cas12a trans-cleavage activity and specific recognition of ZIF-67 to ATP. Exciton energy transfer between CdTe QDs and AuNPs system is firstly constructed as photoelectrochemical (PEC) sensing substrate. Then, the activator DNAs, used to activate CRISPR/Cas12a, are absorbed on the surface of ZIF-67. In the presence of ATP, the activator DNAs are released due to more efficient adsorption of ZIF-67 to ATP. The released activator DNA activates trans-cleavage activity of CRISPR/Cas12a to degrade ssDNA on the electrode, leading to the recovery of photocurrent due to the interrupted energy transfer. Benefiting from the specific recognition of ZIF-67 to ATP and CRISPR/Cas12a-modulated amplification strategy, the sensor is endowed with excellent specificity and high sensitivity.

Augmented microglial endoplasmic reticulum-mitochondria contacts mediate depression-like behavior in mice induced by chronic social defeat stress.

Extracellular ATP (eATP) signaling through the P2X7 receptor pathway is widely believed to trigger NLRP3 inflammasome assembly in microglia, potentially contributing to depression. However, the cellular stress responses of microglia to both eATP and stress itself remain largely unexplored. Mitochondria-associated membranes (MAMs) is a platform facilitating calcium transport between the endoplasmic reticulum (ER) and mitochondria, regulating ER stress responses and mitochondrial homeostasis. This study aims to investigate how MAMs influence microglial reaction and their involvement in the development of depression-like symptoms in response to chronic social defeat stress (CSDS). CSDS induced ER stress, MAMs' modifications, mitochondrial damage, and the formation of the IP3R3-GRP75-VDAC1 complex at the ER-mitochondria interface in hippocampal microglia, all concomitant with depression-like behaviors. Additionally, exposing microglia to eATP to mimic CSDS conditions resulted in analogous outcomes. Furthermore, knocking down GRP75 in BV2 cells impeded ER-mitochondria contact, calcium transfer, ER stress, mitochondrial damage, mitochondrial superoxide production, and NLRP3 inflammasome aggregation induced by eATP. In addition, reduced GRP75 expression in microglia of Cx3cr1CreER/+Hspa9f/+ mice lead to reduce depressive behaviors, decreased NLRP3 inflammasome aggregation, and fewer ER-mitochondria contacts in hippocampal microglia during CSDS. Here, we show the role of MAMs, particularly the formation of a tripartite complex involving IP3R3, GRP75, and VDAC1 within MAMs, in facilitating communication between the ER and mitochondria in microglia, thereby contributing to the development of depression-like phenotypes in male mice.

Assembly of the Tn7 targeting complex by a regulated stepwise process.

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.

Dual pH- and ATP-Responsive Antibacterial Nanospray: On-Demand Release of Antibacterial Factors, Imaging Monitoring, and Accelerated Healing of Bacteria-Infected Wounds under NIR Activation.

The prevalence of pathogenic bacterial infections with high morbidity and mortality poses a widespread challenge to the healthcare system. Therefore, it is imperative to develop nanoformulations capable of adaptively releasing antimicrobial factors and demonstrating multimodal synergistic antimicrobial activity. Herein, an NIR-activated multifunctional synergistic antimicrobial nanospray MXene/ZIF-90@ICG was prepared by incorporating ZIF-90@ICG nanoparticles onto MXene-NH2 nanosheets. MXene/ZIF-90@ICG can on-demand release the antimicrobial factors MXenes, ICG, and Zn2+ in response to variations in pH and ATP levels within the bacterial infection microenvironment. Under NIR radiation, the combination of MXenes, Zn2+, and ICG generated a significant amount of ROS and elevated heat, thereby enhancing the antimicrobial efficacy of PDT and PTT. Meanwhile, NIR excitation could accelerate the further release of ICG and Zn2+, realizing the multimodal synergistic antibacterial effect of PDT/PTT/Zn2+. Notably, introducing MXenes improved the dispersion of the synthesized antimicrobial nanoparticles in aqueous solution, rendering MXene/ZIF-90@ICG a candidate for application as a nanospray. Importantly, MXene/ZIF-90@ICG demonstrated antimicrobial activity and accelerated wound healing in the constructed in vivo subcutaneous Staphylococcus aureus infection model with NIR activation, maintaining a favorable biosafety level. Therefore, MXene/ZIF-90@ICG holds promise as an innovative nanospray for adaptive multimodal synergistic and efficient antibacterial applications with NIR activation.

Cordycepin Triphosphate as a Potential Modulator of Cellular Plasticity in Cancer via cAMP-Dependent Pathways: An In Silico Approach.

Cordycepin, or 3'-deoxyadenosine, is an adenosine analog with a broad spectrum of biological activity. The key structural difference between cordycepin and adenosine lies in the absence of a hydroxyl group at the 3' position of the ribose ring. Upon administration, cordycepin can undergo an enzymatic transformation in specific tissues, forming cordycepin triphosphate. In this study, we conducted a comprehensive analysis of the structural features of cordycepin and its derivatives, contrasting them with endogenous purine-based metabolites using chemoinformatics and bioinformatics tools in addition to molecular dynamics simulations. We tested the hypothesis that cordycepin triphosphate could bind to the active site of the adenylate cyclase enzyme. The outcomes of our molecular dynamics simulations revealed scores that are comparable to, and superior to, those of adenosine triphosphate (ATP), the endogenous ligand. This interaction could reduce the production of cyclic adenosine monophosphate (cAMP) by acting as a pseudo-ATP that lacks a hydroxyl group at the 3' position, essential to carry out nucleotide cyclization. We discuss the implications in the context of the plasticity of cancer and other cells within the tumor microenvironment, such as cancer-associated fibroblast, endothelial, and immune cells. This interaction could awaken antitumor immunity by preventing phenotypic changes in the immune cells driven by sustained cAMP signaling. The last could be an unreported molecular mechanism that helps to explain more details about cordycepin's mechanism of action.

Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase.

Regulatory cystathionine ß-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.

Miniaturizable Chemiluminescence System for ATP Detection in Water.

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

Kinase-catalyzed crosslinking: A comparison of ATP-crosslinker analogs.

Protein phosphorylation is catalyzed by kinases to regulate cellular events and disease states. Identifying kinase-substrate relationships represents a powerful strategy to understand cell biology and disease yet remains challenging due to the rapid dynamics of phosphorylation. Over the last decade, several γ-phosphoryl modified ATP analogs containing crosslinkers were developed to covalently conjugate kinases, their substrates, and their associated proteins for subsequent characterization. Here, kinetics and crosslinking experiments demonstrated that the UV-activated analogs, ATP-aryl azide and ATP-benzophenone, offered the most robust crosslinking, whereas electrophilic ATP-aryl fluorosulfate promoted the most effective proximity-enabled crosslinking. The data will guide future applications of kinase-catalyzed crosslinking to study normal and disease biology.

Aptamer responsive DNA Functionalized hydrogels electrochemiluminescence biosensor for the detection of adenosine triphosphate.

DNA hydrogel represents a noteworthy biomaterial. The preparation of biosensors by combining DNA hydrogel with electrochemiluminescence can simplify the modification process and raise the experimental efficiency. In this study, an electrochemiluminescence (ECL) biosensor based on DNA hydrogel was fabricated to detect adenosine triphosphate (ATP) simply and quickly. CdTe-Ru@SiO2 nanospheres capable of ECL resonance energy transfer (RET) were synthesized and encapsulated CdTe-Ru@SiO2 in the DNA hydrogel to provide strong and stable ECL signals. DNA hydrogel avoided the labeling of ECL signal molecules. The aptamer of ATP as the linker of the hydrogel for the specificity of ATP detection. The cross-linked structure of the aptamer and the polymer chains was opened by ATP, and then the decomposition of the DNA hydrogel initiated the escape of CdTe-Ru@SiO2 to generate an ECL signal. The designed biosensor detected ATP without too much modification and complex experimental steps on the electrode surface, with good specificity and stability, and a wide linear range. The detection range was 10-5000 nM, and the detection limit was 6.68 nM (S/N = 3). The combination of DNA hydrogel and ECL biosensor provided a new way for clinical detection of ATP and other biomolecule.

Neural stem/progenitor cells from olfactory neuroepithelium collected by nasal brushing as a cell model reflecting molecular and cellular dysfunctions in schizophrenia.

OBJECTIVES: Neural stem/progenitor cells derived from olfactory neuroepithelium (hereafter olfactory neural stem/progenitor cells, ONSPCs) are emerging as a potential tool in the exploration of psychiatric disorders. The present study intended to assess whether ONSPCs could help discern individuals with schizophrenia (SZ) from non-schizophrenic (NS) subjects by exploring specific cellular and molecular features. METHODS: ONSPCs were collected from 19 in-patients diagnosed with SZ and 31 NS individuals and propagated in basal medium. Mitochondrial ATP production, expression of ß-catenin and cell proliferation, which are described to be altered in SZ, were examined in freshly isolated or newly thawed ONSPCs after a few culture passages. RESULTS: SZ-ONSPCs exhibited a lower mitochondrial ATP production and insensitivity to agents capable of positively or negatively affecting ß-catenin expression with respect to NS-ONSPCs. As to proliferation, it declined in SZ-ONSPCs as the number of culture passages increased compared to a steady level of growth shown by NS-ONSPCs. CONCLUSIONS: The ease and safety of sample collection as well as the differences observed between NS- and SZ-ONSPCs, may lay the groundwork for a new approach to obtain biological material from a large number of living individuals and gain a better understanding of the mechanisms underlying SZ pathophysiology.