Adenosine Triphosphate Research Articles

A Well‐Coupled Supramolecular System Accelerates Photophosphorylation

AbstractSupramolecular assembly allows multiple chemical/bio‐components integrated as one system for cascade biochemical reactions. Herein the graphitic carbon nitrides (g‐C3N4) as photocatalyst trapped in a dipeptide hydrogel covering adenosine triphosphate (ATP) synthase accelerates the photophosphorylation through ATP synthesis. Self‐assembled N‐fluorenylmethoxycarbonyl diphenylalanine (Fmoc‐FF) as nanofibrils to allow g‐C3N4 nanosheets are embedded as a complex Fmoc‐FF/g‐C3N4 hydrogel. Fmoc‐FF gel exhibits good electronic coupling with g‐C3N4, which enables a photo‐induced proton generation. The transmembrane proton gradient can be established by ATP synthase‐lipid reconstituted on the surface of the Fmoc‐FF/g‐C3N4 hydrogel to enhance the ATP synthesis. It indicates that the Fmoc‐FF/g‐C3N4/ATP synthase‐lipid film can possess a longer‐term ATP production capability and allow repeated immersion for sustained ATP production. Such a hydrogel‐supported ATP synthesis platform is achieved by a procedure at a larger scale.

A Well-Coupled Supramolecular System Accelerates Photophosphorylation.

Supramolecular assembly allows multiple chemical/bio-components integrated as one system for cascade biochemical reactions. Herein the graphitic carbon nitrides (g-C3N4) as photocatalyst trapped in a dipeptide hydrogel covering adenosine triphosphate (ATP) synthase accelerates the photophosphorylation through ATP synthesis. Self-assembled N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) as nanofibrils to allow g-C3N4 nanosheets are embedded as a complex Fmoc-FF/g-C3N4 hydrogel. Fmoc-FF gel exhibits good electronic coupling with g-C3N4, which enables a photo-induced proton generation. The transmembrane proton gradient can be established by ATP synthase-lipid reconstituted on the surface of the Fmoc-FF/g-C3N4 hydrogel to enhance the ATP synthesis. It indicates that the Fmoc-FF/g-C3N4/ATP synthase-lipid film can possess a longer-term ATP production capability and allow repeated immersion for sustained ATP production. Such a hydrogel-supported ATP synthesis platform is achieved by a procedure at a larger scale.

Nitrogen-Rich Molybdenum Nitride with Intrinsic CD39 Nucleotidase Activity.

CD39 is one of the important nucleotidases to adjust extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) concentration. However, the enzyme mimics to simulate the activity of CD39 still remains to be explored. Herein nitrogen-rich molybdenum nitride (Mo5N6) nanosheets are explored to possess CD39-like activity, which are able to catalyze the hydrolysis of the high-energy phosphate bonds (HEPBs) in ATP and ADP but not the common phosphate bonds in adenosine monophosphate (AMP). The catalytic hydrolysis of the phosphate bond over Mo5N6-700 nanosheets is first investigated using para-nitrophenyl phosphate as the model substrate and then the CD39-like activity is further explored and verified by 31p NMR spectroscopy. Mo4+ on the surface of Mo5N6-700 nanosheets are the catalytic active sites. Using ATP as the model substrate, the Km and Vmax values of CD39-like activity at optimal pH 9.0 are 3.2µmol L-1 and 18.5µmol L-1 h-1, respectively. The CD39-like activity of Mo5N6-700 nanosheets enabled the down-regulation of intracellular ATP concentration to a larger degree for cancer cells than normal cells, which makes Mo5N6-700 nanosheets a potential therapeutic reagent for cancers.

Seasonal Variation in ATP-Induced Retinal Damage in the Cone-Dominant 13-Lined Ground Squirrel.

To examine whether time of year (relative to hibernation emergence) influences the retinal degenerative effects of intravitreal injection of adenosine triphosphate (ATP) in the 13-lined ground squirrel (13-LGS). Eighteen (9 male, 9 female) 13-LGS in three experimental cohorts (early season, mid-season, late season) (n = 6 each) underwent baseline imaging using scanning light ophthalmoscopy (SLO) and optical coherence tomography (OCT). Animals then received a 10-µL intravitreal injection of 0.723 M ATP, followed by OCT and SLO imaging at 3, 10, and 21 days. Adaptive optics SLO (AOSLO) was performed in animals without retinal damage after the 21-day follow-up. Retinal thickness, choroidal thickness, and cone density measures were compared to values from wild-type controls (n = 12). Five animals (four early season, one late season) showed retinal damage post-ATP injection (Fisher's exact test, P = 0.065). Animals with retinal damage displayed areas of disrupted retinal lamination on OCT. Any changes in OCT thickness were generally present on initial follow-up and resolved at later time points. Follow-up imaging with AOSLO on animals without retinal damage showed no significant differences in the cone mosaic topography from control eyes. Axial length was increased in mid-/late-season cohorts relative to early season (P = 0.0025 and P = 0.0007). In this pilot study, the 13-LGS appears more susceptible to ATP-induced retinal damage during the early season. Future studies adjusting dose based on ocular biometry may help elucidate the impact of time of year on chemical response. Consideration of ocular biometry in this and other animal models is merited when using intravitreal methods of chemical administration.

Simvastatin Efficacy on Endodontic Biofilms: An In Vitro Study.

Mature biofilms were grown on dentin specimens and put in contact with the solutions to determine their antibiofilm activity. To assess residual antimicrobial activity, the dentin samples were immersed for 5 min in the solutions before microbial infection for 3 weeks. The study groups were: (1) 8% SIM, (2) 4% SIM, (3) 4% DC, (4) 2.5% sodium hypochlorite (NaOCl), and (5) 0.9% saline solution (SS). Cell viability was evaluated by means of the adenosine triphosphate (ATP) assay and flow cytometry (FC). The data collected were analyzed with the ANOVA test using Welch's correction followed by the Games-Howell test. The level of statistical significance was p < 0.05, and statistical analysis was performed using SPSS 23.0. All study groups reduced the biofilms significantly with respect to the control. The highest reduction percentage was obtained by 2.5% NaOCl, followed by SIM 8% and 4%, without statistically significant differences. In terms of residual activity, the 4% DC solution obtained a higher percentage of dead cells. Solutions of 4% and 8% SIM, and 4% Diclofenac, show antimicrobial and residual activity against multispecies endodontic biofilms.

The bioenergetic cost of building a metazoan

All life forms depend on the conversion of energy into biomass used in growth and reproduction. For unicellular heterotrophs, the energetic cost associated with building a cell scales slightly sublinearly with cell weight. However, observations on multiple Daphnia species and numerous other metazoans suggest that although a similar size-specific scaling is retained in multicellular heterotrophs, there is a quantum leap in the energy required to build a replacement soma, presumably owing to the added investment in nonproductive features such as cell adhesion, support tissue, and intercellular communication and transport. Thus, any context-dependent ecological advantages that accompany the evolution of multicellularity come at a high baseline bioenergetic cost. At the phylogenetic level, for both unicellular and multicellular eukaryotes, the energetic expense per unit biomass produced declines with increasing adult size of a species, but there is a countergradient scaling within the developmental trajectories of individual metazoan species, with the cost of biomass production increasing with size. Translation of the results into the universal currency of adenosine triphosphate (ATP) hydrolyses provides insight into the demands on the electron-transport/ATP-synthase machinery per organism and on the minimum doubling times for biomass production imposed by the costs of duplicating the energy-producing infrastructure.

De Novo Biosynthesis of 2-Phenylethanol by Metabolic Engineering the Oleaginous Yeast Rhodotorula toruloides.

Microbial production offers a sustainable route to plant- and chemical-based manufacturing. 2-Phenylethanol (2-PE) has been widely used in foods, flavors, and pharmaceuticals. Herein, de novo biosynthesis of 2-PE was achieved for the first time by rewiring the non-native producer Rhodotorula toruloides. Our results showed that the generation of phenylalanine (Phe) and the recycling of the amino group could be bypassed since the Ehrlich pathway outperformed the phenylacetaldehyde-dependent one in de novo biosynthesis of 2-PE when the adenosine 5'-triphosphate (ATP) citrate synthase (ACL) was inactivated. The 2-PE titer was enhanced to 151.7 mg/L in a shake flask by alleviating feedback inhibition, enhancing precursor availability and cofactor balance. Finally, the production of 2-PE was elevated to 1.06 g/L with a yield of 8.5 mg/g glucose and productivity of 8 mg/L/h in a 3 L bioreactor. Our results should shed light on the microbial production of other aromatic derivatives with R. toruloides.

Simultaneous assessment of cerebral glucose and oxygen metabolism and perfusion in rats using interleaved deuterium (2H) and oxygen-17 (17O) MRS.

Cerebral glucose and oxygen metabolism and blood perfusion play key roles in neuroenergetics and oxidative phosphorylation to produce adenosine triphosphate (ATP) energy molecules in supporting cellular activity and brain function. Their impairments have been linked to numerous brain disorders. This study aimed to develop an in vivo magnetic resonance spectroscopy (MRS) method capable of simultaneously assessing and quantifying the major cerebral metabolic rates of glucose (CMRGlc) and oxygen (CMRO2) consumption, lactate formation (CMRLac), and tricarboxylic acid (TCA) cycle (VTCA); cerebral blood flow (CBF); and oxygen extraction fraction (OEF) via a single dynamic MRS measurement using an interleaved deuterium (2H) and oxygen-17 (17O) MRS approach. We introduced a single-loop multifrequency radio-frequency (RF) surface coil that can be used to acquire proton (1H) magnetic resonance imaging (MRI) or interleaved low-γ X-nuclei 2H and 17O MRS. By combining this RF coil with a modified MRS pulse sequence, 17O-isotope-labeled oxygen gas inhalation, and intravenous 2H-isotope-labeled glucose administration, we demonstrate for the first time the feasibility of simultaneously and quantitatively measuring six important physiological parameters, CMRGlc, CMRO2, CMRLac, VTCA, CBF, and OEF, in rat brains at 16.4T. The interleaved 2H-17O MRS technique should be readily adapted to image and study cerebral energy metabolism and perfusion in healthy and diseased brains.

Bioenergetic suppression by redox-active metabolites promotes antibiotic tolerance in Pseudomonas aeruginosa

The proton-motive force (PMF), consisting of a pH gradient and a membrane potential (ΔΨ) underpins many processes essential to bacterial growth and/or survival. Yet bacteria often enter a bioenergetically diminished state characterized by a low PMF. Consequently, they have increased tolerance for diverse stressors, including clinical antibiotics. Despite the ubiquity of low metabolic rates in the environment, the extent to which bacteria have agency over entry into such a low-bioenergetic state has received relatively little attention. Here, we tested the hypothesis that production of redox-active metabolites (RAMs) could drive such a physiological transition. Pseudomonas aeruginosa is an opportunistic pathogen that produces phenazines, model RAMs that are highly toxic in the presence of molecular oxygen (O2). Under oxic conditions, the phenazines pyocyanin and phenazine-1-carboximide, as well as toxoflavin-a RAM produced by Burkholderia species-suppress the ΔΨ in distinct ways across distributions of single cells, reduce the efficiency of proton pumping, and lower cellular adenosine-triphosphate (ATP) levels. In planktonic culture, the degree and rate by which each RAM lowers the ΔΨ correlates with the protection it confers against antibiotics that strongly impact cellular energy flux. This bioenergetic suppression requires the RAM's presence and corresponds to its cellular reduction rate and abiotic oxidation rate by O2; it can be reversed by increasing the ΔΨ with nigericin. RAMs similarly impact the bioenergetic state of cells in (hyp)oxic biofilm aggregates. Collectively, these findings demonstrate that bacteria can suppress their bioenergetic state by the production of endogenous toxins in a manner that bolsters stress resilience.

The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative breast cancer

BackgroundBreast cancer, one of the most common forms of cancer, is associated with the highest cancer-related mortality among women worldwide. In comparison to other types of breast cancer, patients diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst outcome because current therapies do not produce long-lasting responses. Hence, innovative therapies that produce persisting responses are a critical need. We previously discovered that hyperactivating purinergic receptors (P2RXs) by increasing extracellular adenosine triphosphate (eATP) concentrations enhances TNBC cell lines’ response to chemotherapy. Heparan sulfate inhibits multiple extracellular ATPases, so it is a molecule of interest in this regard. In turn, heparanase degrades polysulfated polysaccharide heparan sulfate. Importantly, previous work suggests that breast cancer and other cancers express heparanase at high levels. Hence, as heparan sulfate can inhibit extracellular ATPases to facilitate eATP accumulation, it may intensify responses to chemotherapy. We postulated that heparanase inhibitors would exacerbate chemotherapy-induced decreases in TNBC cell viability by increasing heparan sulfate in the cellular microenvironment and hence, augmenting eATP.MethodsWe treated TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells with paclitaxel (cytotoxic chemotherapeutic) with or without the heparanase inhibitor OGT 2115 and/or supplemental heparan sulfate. We evaluated cell viability and the release of eATP. Also, we compared the expression of heparanase protein in cell lines and tissues by immunoblot and immunohistochemistry, respectively. In addition, we examined breast-cancer-initiating cell populations using tumorsphere formation efficiency assays on treated cells.ResultsWe found that combining heparanase inhibitor OGT 2115 with chemotherapy decreased TNBC cell viability and tumorsphere formation through increases in eATP and activation of purinergic receptors as compared to TNBC cells treated with single-agent paclitaxel.ConclusionOur data shows that by preventing heparan sulfate breakdown, heparanase inhibitors make TNBC cells more susceptible to chemotherapy by enhancing eATP concentrations.

Re-Examination of the Sel’kov Model of Glycolysis and Its Symmetry-Breaking Instability Due to the Impact of Diffusion with Implications for Cancer Imitation Caused by the Warburg Effect

We revisit the seminal model of glycolysis first proposed by Sel’kov more than fifty years ago. We investigate the onset of instabilities in biological systems described by the Sel’kov model in order to determine the conditions of the model parameters that lead to bifurcations. We analyze the glycolysis reaction under the circumstances when the diffusivity of both ATP and ADP reactants are taken into account. We estimate the critical value of the model’s single compact dimensionless parameter, which is responsible for the onset of reaction instability and the system’s symmetry breaking. It appears that it leads to spatial inhomogeneities of reactants, leading to the formation of standing waves instead of a homogeneous distribution of ATP molecules. The consequences of this model and its results are discussed in the context of the Warburg effect, which signifies a transition from oxidative phosphorylation to glycolysis that is correlated with the initiation and progression of cancer. Our analysis may lead to the selection of therapeutic interventions in order to prevent the symmetry-breaking phenomenon described in our work.

Evaluating ATP testing for distribution system monitoring: comparison to HPC, impact of chlorine quenching, and hold time dependency.

Adenosine triphosphate (ATP) assays have a faster turnaround time and higher sensitivity than traditional cultivation methods for microbial monitoring. Challenges implementing ATP testing include incompatibility with chlorine quenching agents and hold time sensitivity, which are not well-studied. Chlorinated distribution system samples were collected from two Canadian utilities, Metro Vancouver (n = 40 samples) and Halifax Water (n = 283). No significant correlations were observed between heterotrophic cell count (HPC) and cellular ATP, suggesting these do not correlate well in waters with low biological activity (median HPC < 2CFU/mL). However, interpretation of HPC and cATP results (based on the HPC guideline of 100CFU/mL and cATP of 10pg/mL) yielded the same conclusion for 95% of samples, suggesting a potential decision-making framework to replace HPC with cATP. Moreover, cATP correlates better with free chlorine (p < 0.04) compared with HPC for one of the studied systems. Importantly, adding chlorine quench (10% sodium thiosulfate) did not produce significantly different cATP results, nor did analyzing at various hold times of 4-, 6-, and 24-h. This study supports the integration of ATP testing into existing sampling procedures for water utilities, as a sensitive, fast, and reliable monitoring method.

Irisin Attenuates Hepatic Stellate Cell Activation and Liver Fibrosis in Bile Duct Ligation Mice Model and Improves Mitochondrial Dysfunction.

Liver fibrosis is a common outcome of chronic liver disease and is primarily driven by hepatic stellate cell (HSC) activation. Irisin, a myokine released during physical exercise, is beneficial for metabolic disorders and mitochondrial dysfunction. This study aimed to explore the effects of irisin on liver fibrosis in HSCs, a bile duct ligation (BDL) mouse model, and the associated mitochondrial dysfunction. In vitro experiments utilized LX-2 cells, a human HSC line, stimulated with transforming growth factor-β1 (TGF-β1), a major regulator of HSC fibrosis, with or without irisin. Mitochondrial function was assessed using mitochondrial fission markers, transmission electron microscopy, mitochondrial membrane potential, and adenosine triphosphate (ATP) production. In vivo, liver fibrosis was induced in mice via BDL, followed by daily intraperitoneal injections of irisin (100 μg/kg/day) for 10 days. In vitro, irisin mitigated HSC activation and reduced reactive oxygen species associated with the TGF-β1/Smad signaling pathway. Irisin restored TGF-β1-induced increases in fission markers (Fis1, p-DRP1) and reversed the decreased expression of TFAM and SIRT3. Additionally, irisin restored mitochondrial membrane potential and ATP production lowered by TGF-β1 treatment. In vivo, irisin ameliorated the elevated liver-to-body weight ratio induced by BDL and alleviated liver fibrosis, as evidenced by Masson's trichrome staining. Irisin also improved mitochondrial dysfunction induced by BDL surgery. Irisin effectively attenuated HSC activation, ameliorated liver fibrosis in BDL mice, and improved associated mitochondrial dysfunction. These findings highlight the therapeutic potential of irisin for the treatment of liver fibrosis.

Microglia modulate cerebral blood flow and neurovascular coupling through ectonucleotidase CD39.

Microglia and the border-associated macrophages (BAMs) contribute to the modulation of cerebral blood flow (CBF), but the mechanisms have remained ill-defined. Here, we show that microglia regulate the CBF baseline and upsurges after whisker stimulation or intracisternal magna injection of adenosine triphosphate (ATP). Genetic or pharmacological depletion of microglia reduces the activity-dependent hyperemia but not the cerebrovascular responses to adenosine stimulation. Notably, microglia repopulation corrects these CBF reactivity deficits. The microglial-dependent regulation of CBF requires the ATP-sensing P2ry12 receptor and the ectonucleotidase CD39 that initiates the breakdown of extracellular ATP. Pharmacological inhibition or microglia-specific deletion of CD39 simulates the CBF anomalies detected in microglia-deficient mice and reduces the rise of extracellular adenosine after whisker stimulation. Together, these results suggest that the microglial CD39-initiated conversion of extracellular ATP to adenosine is an important step in neurovascular coupling and the regulation of cerebrovascular reactivity.

Molecular dynamics simulations provide insights into ULK-101 potency and selectivity toward autophagic kinases ULK1/2

Kinase domains are highly conserved within proteins in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. ULK1 and ULK2 are serine/threonine kinases that serve important roles in autophagy, an intracellular recycling process capable of degrading proteins and organelles via fusion with lysosomes. ULK1/2 are emerging as therapeutic targets in human cancer, particularly KRAS-driven malignancies. Here, we performed molecular dynamics (MD) simulations to hypothesize bound poses for the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the αC-helix. Large movements in the phosphate-binding loop (P-loop) are also associated with ULK-101 inhibitor binding and exit from ULK2. Together, our data support a model to explain ULK-101 potency toward ULK1/2.

Cryo-EM structures of ABCC1 revealing new conformational dynamics in the resting state

ABCC1/MRP1 in the C branch of Adenosine triphosphate binding cassette (ABC) transporters superfamily, is directly linked to multiple drug resistance in chemotherapy. Here, to further understand the conformational dynamics of ABCC1, we performed single-particle cryo-electron microscopy analysis of purified bovine ABCC1. Two conformational states were found coexisted with nearly equal population. While one state has a wider substrate transporting pathway, akin to the previously reported apo structure, the other is narrower, despite the empty substrate pocket. In addition, multiple lipid-binding interfaces were identified based on the presence of rod-shaped, unmodeled, non-protein densities in the resolved density maps, potentially contributing to the stabilization of TMD0 domain and activity regulation of ABCC1. Further, we found that three asparagine residues in bovine ABCC1 are glycosylated. Together, our study provides fresh insights into the structural features and conformational dynamics of bovine ABCC1, offering a new framework for understanding the function and regulatory mechanisms of ABCC1.

Insights into the antifungal activity and mechanisms of cinnamon components against Aspergillus flavus and Penicillium citrinum

Fungal spoilage of food and the excessive use of chemical disinfectants serves potential adverse effects on human health and the environment. Consequently, there is a growing interest in exploring natural alternatives, particularly plant-derived antimicrobial preservatives. Cinnamon extracts are known for their antifungal activity, but most research has focused on essential oils, rarely on other bioactive components. This study assessed the antifungal activity and underlying mechanisms of four components—trans-cinnamaldehyde, cis-2-methoxycinnamic acid, coumarin, and o-methoxycinnamaldehyde—extracted from Cinnamomum cassia Presl (cinnamon) against Aspergillus flavus and Penicillium citrinum. These cinnamon components can inhibit the two fungi strains at the minimum inhibitory concentration ranged from 0.30 to 8.55 mmol/L. These components can disrupt fungal cell membranes by enhancing relative electrical conductivity and cytoplasmic content leakage, reducing ergosterol content, and increasing malondialdehyde level. Additionally, they can affect fungal cell wall integrity, leading to the leakage of alkaline phosphatase and alterations in the contents of β-1,3-glucan and chitin. Moreover, the cinnamon components influenced the activities of malate dehydrogenase, succinate dehydrogenase, as well as adenosine triphosphate levels. The observed suppression of fungal contamination in A. flavus and P. citrinum suggests that these cinnamon components as potential natural antifungal agents.

Bacillus amyloliquefaciens SC06 alleviates LPS-induced intestinal damage by inhibiting endoplasmic reticulum stress and mitochondrial dysfunction in piglets

Endoplasmic reticulum stress (ERS) and mitochondrial dysfunction play an important role in the pathogenesis of intestinal diseases. Our studies investigated the effects of Bacillus amyloliquefaciens SC06 on jejunal mitochondria and ER in piglets under the LPS-induced intestinal injury model. Eighteen piglets (male, 21 days old) were randomly assigned to three treatments: CON (basal diet), LPS (basal diet +100 μg/kg LPS), and SC06 + LPS (basal diet +1 × 108 cfu/kg SC06 + 100 μg/kg LPS). Compared to the LPS group, administration of SC06 improved jejunal morphology and barrier function. In addition, SC06 reduced reactive oxygen species (ROS) and MDA generation in the jejunum by activating the Nrf2/keap1 pathway, which increased the activity of CAT, GSH and SOD in LPS-challenged pigs. In addition, SC06 reduced LPS-induced mitochondrial dysfunction and ERS as evidenced by a decrease in ROS, an improvement in mitochondrial membrane potential and an increase in adenosine triphosphate levels. The results of in vitro IPEC-J2 cell experiments also indicate that SC06 can reduce LPS-induced oxidative stress, mitochondrial dysfunction, ERS, and intestinal barrier function damage by activating the Nrf2/keap1 signaling pathway. Finally, treatment with the Nrf2-specific inhibitor ML-385 inhibited the upregulated effect of SC06 on antioxidant capacity and intestinal barrier function in IPEC-J2 cells. In conclusion, SC06 ameliorated intestinal damage and mitochondrial dysfunction and attenuated endoplasmic reticulum stress via activation of the Nrf2/keap1 signaling pathway in LPS-challenged piglets.

Hexagonal Prism-Shaped AIE-Active MOFs as Coreactant-Free Electrochemiluminescence Luminophores Coupled with Hollow Cu2-xO@Pd Heterostructures as Efficient Quenching Probes for Sensitive Biosensing.

For most self-luminous metal-organic framework (MOF)-involved electrochemiluminescence (ECL) systems, the integration of exogenous coreactants is indispensable to promote ECL efficiency. However, the introduction of a coreactant into an electrolyte would result in poor stability, thereby inevitably affecting analytical accuracy. Herein, by employing aggregation-induced emission luminogens as ligands, we first synthesized one hexagonal prism-shaped MOF that displays robust and steady ECL signal without an exogenous coreactant. Furthermore, adenosine triphosphate (ATP), as the target analyte, can be fixed on the electrode surface directly owing to the strong coordination between Zr4+ and phosphate groups. According to the ECL resonance energy transfer effect, hollow Cu2-xO@Pd heterostructures are conveniently prepared and act as efficient quenching probes. Remarkably, the resultant urchin-like hollow structure could provide more active sites to anchor ATP aptamers, thus enhancing the ECL quenching efficiency. In this manner, an elaborate coreactant-free ECL system was developed to detect ATP, which demonstrates a remarkable detection limit of 0.17 nM, as well as excellent stability and reproducibility. The present work offers significant enlightenment for the further evolution of advanced ECL systems integrated with MOF-based luminophores.

Development and validation of an LC‒MS/MS method for the determination of cyclocreatine phosphate and its related endogenous biomolecules in rat heart tissues

The cardioprotective drug cyclocreatine phosphate has been awarded Food and Drug Administration-orphan drug designation for the prevention of ischemic injury to enhance cardiac graft recovery and survival in heart transplantation. Cyclocreatine phosphate is the water-soluble derivative of cyclocreatine. Estimating the levels of Cyclocreatine phosphate, Adenosine triphosphate, Creatine Phosphate, Creatine and Cyclocreatine helps us in understanding the energy state as well as evaluating the heart cells’ function. The quantification of endogenous compounds imposes a challenging task for analysts because of the absence of a true blank matrix, whose use is required according to international guidelines. Recently, the International Council for Harmonization issued a new guideline that contains guidance on the validation of methods used to quantify endogenous components, such as the background subtraction approach that was employed in our current study. Specifically, we developed and validated a sensitive, reliable and accurate liquid chromatography-tandem mass spectrometry assay to determine simultaneously the levels of mentioned endogenous compounds in rat heart tissue. Tissue samples were prepared by protein precipitation extraction using water: methanol (1:1). Using Ultra Performance Liquid Chromatography, Chromatographic separation was achieved with ZORBAX Eclipse Plus C18 4.6 × 100 mm,3.5 μm column and conditions as following: ammonium acetate (pH 8.5): acetonitrile, 70:30 mobile phase, 0.7 mL/min flow rate and 25 °C temperature. Electrospray ionization mass detector with Multiple reaction monitoring mode was then employed, using both positive and negative modes, Analysis was carried out using 5.00–2000.00 ng/mL linear concentration range within 2 min for each analyte. According to Food and Drug Administration guidelines for bioanalytical methods, validation was carried out. We investigated the matrix effect, recovery efficiency and process efficiency for the analyte in neat solvent, postextraction matrix and tissue. The results stated mean percentage recoveries higher than 99%, accuracy 93.32–111.99%, and Relative Standard Deviation (RSD) below 15% within the concentration range of our study which indicated that target analytes’ stability in their real matrix is sufficient under the employed experimental conditions.