Intracellular Adenosine Research Articles

ATP and Adenosine Metabolism in Cancer: Exploitation for Therapeutic Gain.

Adenosine is an evolutionary ancient metabolic regulator linking energy state to physiologic processes, including immunomodulation and cell proliferation. Tumors create an adenosine-rich immunosuppressive microenvironment through the increased release of ATP from dying and stressed cells and its ectoenzymatic conversion into adenosine. Therefore, the adenosine pathway becomes an important therapeutic target to improve the effectiveness of immune therapies. Prior research has focused largely on the two major ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase 1/cluster of differentiation (CD)39 and ecto-5'-nucleotidase/CD73, which catalyze the breakdown of extracellular ATP into adenosine, and on the subsequent activation of different subtypes of adenosine receptors with mixed findings of antitumor and protumor effects. New findings, needed for more effective therapeutic approaches, require consideration of redundant pathways controlling intratumoral adenosine levels, including the alternative NAD-inactivating pathway through the CD38-ectonucleotide pyrophosphatase phosphodiesterase (ENPP)1-CD73 axis, the counteracting ATP-regenerating ectoenzymatic pathway, and cellular adenosine uptake and its phosphorylation by adenosine kinase. This review provides a holistic view of extracellular and intracellular adenosine metabolism as an integrated complex network and summarizes recent data on the underlying mechanisms through which adenosine and its precursors ATP and ADP control cancer immunosurveillance, tumor angiogenesis, lymphangiogenesis, cancer-associated thrombosis, blood flow, and tumor perfusion. Special attention is given to differences and commonalities in the purinome of different cancers, heterogeneity of the tumor microenvironment, subcellular compartmentalization of the adenosine system, and novel roles of purine-converting enzymes as targets for cancer therapy. SIGNIFICANCE STATEMENT: The discovery of the role of adenosine as immune checkpoint regulator in cancer has led to the development of novel therapeutic strategies targeting extracellular adenosine metabolism and signaling in multiple clinical trials and preclinical models. Here we identify major gaps in knowledge that need to be filled to improve the therapeutic gain from agents targeting key components of the adenosine metabolic network and, on this basis, provide a holistic view of the cancer purinome as a complex and integrated network.

Black carrot extract protects against hepatic injury through epigenetic modifications.

We studied the epigenetic regulation of how black carrot extract (BCE) protects against ethanol-induced hepatic damage. We have shown that the butanol-extracted fraction of BCE (BCE-BuOH) increased intracellular cyclic adenosine monophosphate (cAMP) levels by suppressing the expression of phosphodiesterase 4b (PDE4b); however, the detailed mechanism remains to be elucidated. We focused on changes in histone modifications involved in the suppression of pde4 expression. The methylation level of histone H3 lysine 9 (H3K9), which regulates gene expression of PDE4b, decreased after treatment with 100 mM ethanol but was significantly increased by treatment with 400 μg/ml BCE-BuOH. In contrast, ethanol induced an increase in H3K9 acetylation. However, treatment with BCE-BuOH inhibited the increase in acetylation through an increase in Sirtuin 1 (Sirt1), a histone deacetylase. Furthermore, BCE-BuOH treatment increased the level of methionine adenosyltransferase (MAT) 2a mRNA and increased intracellular S-adenosylmethionine. The present results indicate that BCE-BuOH is useful for protection against alcohol-induced hepatic injury. PRACTICAL APPLICATIONS: We have reported that black carrot extract (BCE) suppressed liver steatosis and liver fibrosis on a rat alcoholic liver disease model. The results from this study have shown that BCE regulated the alcoholic-induced hepatic injury at the level of epigenetic modifications. These results suggested that BCE is useful for protection against alcoholic-induced hepatic injury.

Calcium Electroporation Reduces Viability and Proliferation Capacity of Four Uveal Melanoma Cell Lines in 2D and 3D Cultures.

Simple SummaryCalcium electroporation (CaEP) is an innovative anti-tumor treatment modality that induces cell death by introducing supraphysiological concentrations of calcium into cells with a limited effect on normal cells. The objective of the present study is to assess the effect of CaEP in uveal melanoma (UM) cell lines in comparison to electrochemotherapy (ECT) with bleomycin using 2D monolayer cell cultures as well as 3D tumor spheroid models in four different UM cell lines. The morphological changes of the spheroids, the cell viability, growth rate as well as the cytotoxic effect of electroporation (EP) with calcium chloride and bleomycin were evaluated with various drug concentrations. The results of CaEP and ECT both suggest a comparable dose-dependent reduction in cell viability and proliferation rate in all tested 2D cell lines and 3D tumor spheroids. These data point out that CaEP is an established anticancer treatment causing cell death by ATP depletion in in vitro and in vivo, representing an efficient alternative therapy with a lower cytotoxic potency for the local UM tumor control.Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non-cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye.

PGE2 inhibits neutrophil phagocytosis through the EP2R-cAMP-PTEN pathway.

Prostaglandin E2 (PGE2) is a potent lipid mediator of inflammation that modulates immune cell function by binding to unique G protein‐coupled receptors (EP receptors). PGE2 production increases during microbial infection and inflammation. In this study, we assessed the effect of PGE2 on the phagocytosis of bacteria by neutrophils, which are key players during infection and inflammation. We also looked for specific EP receptor signaling pathways that contributed to the neutrophil phagocytic activity. PGE2 (50–1000 ng/ml) inhibited the phagocytosis of Escherichia coli by HL‐60 human neutrophils in a concentration‐dependent manner. Inhibition of neutrophil phagocytosis by PGE2 correlated with increased intracellular cyclic adenosine monophosphate (cAMP) production, and forskolin, an adenosyl cyclase agonist, confirmed the inhibitory effect of cAMP stimulation on neutrophil phagocytosis. The expression of EP2 receptors by HL‐60 cells was confirmed by western blot analysis, and selective agonism of EP2 receptors mimicked the inhibition of phagocytosis by PGE2. The EP2 receptor antagonist AH‐6089 partially blocked the inhibition of neutrophil phagocytosis PGE2. Specific inhibition of phosphatase and tensin homolog (PTEN) enzyme attenuated the inhibition of neutrophil phagocytosis by PGE2, and both PGE2 and increased intracellular cAMP increased neutrophil PTEN activity, which was associated with decreased PTEN phosphorylation. The results support negative regulation of the antimicrobial activity of neutrophils (i.e., phagocytosis), which has important implications for the future management of bacterial infections.

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells.

The GPR15 receptor is a G protein‐coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25–81) and a C‐terminal peptide fragment GPR15L(71–81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3′,5′‐monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs, Gq/11 and G12/13 is unclear. Here, GPR15L(25–81) and GPR15L(71–81) are used as pharmacological tool compounds to delineate the GPR15 receptor‐mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1, Gi2, Gi3, GoA, GoB and Gz). The Emax and activation rates of Gi1, Gi2, Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin.

Effect of 2-methoxyestradiol nanoparticles combined with propofol on hippocampal nerve cells and metabolism after cerebral ischemia-reperfusion

Short-term or permanent reduction in cerebral blood flow can lead to cerebral ischemia, and if it persists, it can lead to necrosis and cell death. Reperfusion is restoration of blood supply, but it may further aggravate the damage of ischemic brain tissue. Propofol has characteristics such as rapid recovery, continuous infusion, and no accumulation. 2-Methoxyestradiol (2ME2) is a HIF-1-α inhibitor and reduces cell apoptosis. The brain ischemia-reperfusion hippocampal nerve cell model was in this study prepared in vitro. Samples were randomly assigned into control group (control), cerebral ischemia-reperfusion (CIR) group, propofol group (Propofol), 2ME2 nanoparticle group (2ME2-NPs), and combination group (Propofol+2ME2-NPs). The changes of cell proliferation and apoptosis activities were analyzed in each group, which included changes of Survivin expression, changes of intracellular Adenosine monophosphate, diphosphate, and triphosphate (AMP, ADP, and ATP, respectively) changes of malondialdehyde (MDA) and superoxide dismutase (SOD) Analysis also included secretion of IL-6 and TNF-α, and detection of HIF-1α and NF-κB expression. Results revealed that, CIR hippocampal nerve cell proliferation decreased, apoptosis activity increased, while Survivin expression increased. Moreover, ATP, ADP and AMP contents decreased, while secretion of inflammatory factors IL-6 and TNF-α increased, MDA increased, and SOD decreased. HIF-1α and NF-κB expressions increased. Propofol and 2ME2 nanoparticles promoted hippocampal neurons proliferation after CIR brain injury, inhibit the apoptotic activity, and reduce the expression of apoptotic protein Survivin. ATP, ADP and AMP content increased, IL-6 and TNF-α decreased, MDA decreased, SOD activity increased, HIF-1α and NF-κB decreased. The and combined group had a more significant effect. 2ME2 nanoparticles combined with propofol can inhibit HIF-1α and secretion of inflammatory factors by regulating oxidative stress and NF-κB signaling pathways, promoting the proliferation of hippocampal neurons, inhibiting apoptosis, and restoring cell energy and helping to restore nerve cell function.

The inhibitory effects of Dulaglutide on cellular senescence against high glucose in human retinal endothelial cells

Diabetic nephropathy is one of the most important chronic microvascular complications of diabetes, and its main feature is diabetic glomerulosclerosis. Endothelial sirtuin 1 (SIRT1) expression is related to aging, and reducing SIRT1 expression promotes endothelial cell aging. Plasminogen activator inhibitor-1 (PAI-1) can be synthesized in a variety of cells, such as endothelial cells. Dulaglutide is a glucagon-like peptide-1 (GLP-1) drug, and it can activate the GLP-1 receptor and promote the conversion of intracellular adenosine triphosphate to adenylate cyclase, thereby activating phosphokinase A, and regulating blood glucose levels effectively in the body. We analyzed the effects of Dulaglutide on inhibiting cell senescence by studying the effects of its different concentrations on telomerase activity and senescence-related gene expression. Our results suggest that Dulaglutide can alleviate high-glucose-induced oxidative stress in human retinal endothelial cells by restoring the expressions of SIRT1 and endothelial nitric oxide synthase (eNOS), thereby inhibiting the expression of PAI-1, and restoring telomerase activity. This suggests that the activity of retinal endothelial cells can be controlled by regulating the expression of SIRT1, so as to achieve the effect of treating diabetic retinopathy.

The Structural Role of the Beta‐2 Adrenergic Receptor Function for the Pharmacological Treatment of Cardiopulmonary Diseases

As a part of the group of Beta‐Adrenergic receptors (B1, B2, B3), the Beta‐2 Adrenergic receptor is a transmembrane glycoprotein that is principally responsible for the mediation of physiological function over the pulmonary and cardiovascular systems through the binding to the catecholamine, adrenaline (epinephrine). Residing in a larger superfamily of hormone‐activated G‐Protein‐Coupled‐Receptors (GPCR), the B2AR adrenoceptor is composed of 3 extracellular and 5 intracellular alpha helices that are coupled with the heterotrimeric G protein, Gs. When the alpha subunit of Gs is bound to GDP, the B2AR adrenoceptor undergoes structural changes upon activation to transmit the communicative signals to the adenylyl cyclase via the stimulatory G‐protein. Consequently, the B2AR receptor resides in two structural conformations: the active and inactive state, dependent on the binding of the GDP to Gs. The shift in equilibrium between each state in proportion to the strength of the binding agonist has brought about new therapeutic research proposing the in silico screening of new B2AR ligands to improve affinity and selectivity profiles of adrenergic agonists. When bound to a full agonist, B2AR exhibits an 11‐Armstrong (A) protrusion of TM6 and inward folding of TM7 and TM3 to transform into a highly unstable arrangement that cannot be adequately established by the potential binding of a stronger agonist alone. The differences inside the ligand‐binding pocket are more subtle but exemplify the weakening of the polar interactions between the amino acids Asn293, Ser203, and Ser207. Once B2AR is activated by any partial or positive agonist, the detached stimulatory G‐protein catalyzes the activity of adenylyl cyclase. In turn, the intracellular cyclic adenosine monophosphate (cAMP) content is elevated significantly. cAMP initiates simultaneous pathways that aid in the release of constricted airways of bronchospastic patients. The pathways induced by the B2AR receptor therapeutically target muscle cell contraction, uterine relaxation, and premature labor within tocolytic drug candidates. Alternatively, B2AR functioning can be restrained by antagonists to inhibit basal, agonist‐independent activity that can be fatally detrimental, such as the continual production of glucose from glycogenolysis. The methodological advances to discover ligands that can function as inverse agonists through the crystallogenesis of B2AR proves to create promising drug treatments for cardiopulmonary diseases involved with muscle contraction. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of B2AR to further investigate the structure of beta‐2‐adrenergic receptor function relative to adrenaline for pharmacological drug discovery.

A raising dawn of pentoxifylline in management of inflammatory disorders in Covid-19.

The existing pandemic viral infection caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) leads to coronavirus disease 2019 (Covid-19). SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as an entry-point into affected cells and down-regulation of ACE2 by this virus triggers the release of pro-inflammatory cytokines and up-regulation of angiotensin II. These changes may lead to hypercytokinemia and the development of cytokine storm with the development of acute lung injury and acute respiratory distress syndrome. Different repurposed had been in use in the management of Covid-19, one of these agents is pentoxifylline (PTX) which has anti-inflammatory and antioxidant properties. Therefore, the objective of the present mini-review is to highlight the potential role of PTX in Covid-19 regarding its anti-inflammatory and antioxidant effects. PTX is a non-selective phosphodiesterase inhibitor that increases intracellular cyclic adenosine monophosphate which stimulates protein kinase A and inhibits leukotriene and tumor necrosis factor. PTX has antiviral, anti-inflammatory and immunomodulatory effects, thus it may attenuate SARS-CoV-2-induced hyperinflammation and related complications. As well, PTX can reduce hyper-viscosity and coagulopathy in Covid-19 through increasing red blood cell deformability and inhibition of platelet aggregations. In conclusion, PTX is a non-selective phosphodiesterase drug, that has anti-inflammatory and antioxidant effects thereby can reduce SARS-CoV-2 infection-hyperinflammation and oxidative stress. Besides, PTX improves red blood cells (RBCs) deformability and reduces blood viscosity so can mitigate Covid-19-induced hyper-viscosity and RBCs hyper-aggregation which is linked with the development of coagulopathy. Taken together, PTX seems to be an effective agent against Covid-19 severity.

Modular Assembly of a Concatenated DNA Circuit for In Vivo Amplified Aptasensing.

Probing endogenous molecular profiles in living entities is of fundamental significance to decipher biological functions and exploit novel theranostics. Despite programmable nucleic acid-based aptasensing systems across the breadth of molecular imaging, an aptasensing system enabling in vivo imaging with high sensitivity, accuracy, and adaptability is highly required yet is still in its infancy. Artificial catalytic DNA circuits that can modularly integrate to generate multiple outputs from a single input in an isothermal autonomous manner, have supplemented powerful toolkits for intracellular biosensing research. Herein, a multilayer nonenzymatic catalytic DNA circuits-based aptasensing system is devised for in situ imaging of a bioactive molecule in living mice by assembling branched DNA copolymers with high-molecular-weight and high-signal-gain based on avalanche-mimicking hybridization chain reactions (HCRs). The HCRs aptasensing circuit performs as a general and powerful sensing platform for precise analysis of a series of bioactive molecules due to its inherent rich recognition repertoire and hierarchical reaction accelerations. With tumor-targeting capsule encapsulation, the HCRs aptasensing circuit is specifically delivered into tumor cells and allowed the high-contrast imaging of intracellular adenosine triphosphate in living mice, highlighting its potential for visualizing these clinically important biomolecules and for studying the associated physiological processes.

Validation of a therapeutic strategy involving the mitochondrial delivery of thiamine pyrophosphate using brain damage induced mouse model

AbstractIschemic brain disease, a type of cerebrovascular disease, has a high mortality rate, and even when patients survive, they are frequently affected by neurological dysfunction due to damage to brain cells. Strategies to increase intracellular adenosine triphosphate (ATP) production are promising approaches for tissue regeneration and restoring neurological function. We report here on an attempt to deliver thiamine pyrophosphate (TPP), a coenzyme that activates the tricarboxylic acid cycle, which is responsible for ATP production, to mitochondria. To achieve this, we used a MITO‐Porter, a liposomal drug delivery system for delivering various cargoes to mitochondria via membrane fusion. In the present study, we investigated the therapeutic effect of the MITO‐Porter (TPP) focusing on its delivery to the brain and neurological function using a bilateral common carotid artery occlusion (BCCAO) mouse model. Fluorescent microscopic analyses confirmed, after a cerebral ischemia‐reperfusion injury had been induced, that the intracerebral delivery of the MITO‐Porter occurred after intravenous administration. Behavioral analyses indicated that the MITO‐Porter (TPP) resulted in significant therapeutic effects compared to the corresponding administration of naked TPP. Histological evaluations also confirmed that the MITO‐Porter (TPP) clearly had an effect on the growth of brain neurons in the BCCAO mouse. Our findings indicate that the mitochondrial delivery of TPP represents a potential innovative therapeutic strategy for enhancing mitochondrial energy production in certain cases of brain diseases.

A novel NCI-H69V small cell lung cancer functional mini-tumor model for future treatment screening applications.

Small cell lung cancer (SCLC) is aggressive and despite multiple clinical trials, its standard of care is unchanged for the past three decades. In vitro cancer models are crucial in chemotherapy development, and three-dimensional (3D) models aim to bridge the gap between two-dimensional (2D) flat cultures and in vivo testing. Functional 3D spheroids can better represent the in vivo situation and tumor characteristics than 2D models. An NCI-H69V SCLC mini-tumor model was developed in a clinostat-based rotating bioreactor system. Spheroid growth and viability were characterized for 30 days, and the ideal experimental window with mature and metabolically stable spheroids was determined. Application of the model for anticancer treatment screening was validated with the standard chemotherapeutic drug irinotecan, for an exposure period of 72 h. The following parameters were measured: soluble protein content, planar surface area measurements, intracellular adenosine triphosphate and extracellular adenylate kinase levels, and glucose consumption. Histological morphology of the spheroids was observed. The established model proved viable and stable, while treatment with irinotecan caused a decrease in cell growth, viability, and glucose consumption demonstrating reactivity of the model to chemotherapy. Therefore, this NCI-H69V SCLC functional spheroid model could be used for future anticancer compound screening.

Novel Opioid Analgesics for the Development of Transdermal Opioid Patches That Possess Morphine-Like Pharmacological Profiles Rather Than Fentanyl: Possible Opioid Switching Alternatives Among Patch Formula.

BACKGROUND:Transdermal fentanyl is widely used in the treatment of severe pain because of convenience, safety, and stable blood concentrations. Nevertheless, patients often develop tolerance to fentanyl, necessitating the use of other opioids; transdermal buprenorphine patch is widely used as an analgesic agent, though available formulation does not provide comparable analgesic effect as transdermal fentanyl patch. Opioids bind to the opioid receptor (OR) to activate both G protein–mediated and β-arrestin–mediated pathways. We synthesized morphine-related compounds with high transdermal absorbability (N1 and N2) and evaluated their OR activities pharmacologically in comparison with fentanyl and morphine.METHODS:In cells stably expressing μ-opioid receptor (MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR), G protein–mediated pathways were assessed using the CellKey and an intracellular cyclic adenosine monophosphate (cAMP) assay, while β-arrestin–mediated pathways were analyzed with β-arrestin recruitment and receptor internalization assays. Furthermore, analgesic effects were evaluated using a tail-flick test in mice, and the analgesic effect on fentanyl-tolerant mice was evaluated.RESULTS:In the CellKey and cAMP assays, both N1 and N2 showed the highest affinity for MOR and acted as full agonists as well as partial agonists for DOR and KOR. In the β-arrestin and internalization assays, only fentanyl acted as a full agonist; N1 and N2 acted as partial agonists of MOR. In the mouse tail-flick test, N1 and N2 showed analgesic effects equivalent to those of fentanyl and morphine. In fentanyl-tolerant mice, fentanyl showed a diminished analgesic effect, whereas N1 and N2 as well as morphine retained their analgesic effects.CONCLUSIONS:While N1 and N2 have higher transdermal absorbability than fentanyl, they also have analgesic effects comparable to those of morphine, suggesting that they may be attractive compounds for the development of novel opioid patches for transitioning from fentanyl patches.

Antibacterial Effect of Chrysanthemum Buds' Crude Extract Against Salmonella Typhimurium and Potential Application in Cooked Chicken.

The objective of this study was to clarify the antibacterial activity and mechanism of Chrysanthemum buds' crude extract (CBCE) against Salmonella Typhimurium, and explore the potential application in cooked chicken. The zone of inhibition (ZI), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were used to assess the in vitro antibacterial activity of CBCE against Salmonella Typhimurium. The antibacterial mechanism was elucidated by revealing the changes in intracellular adenosine 5'-triphosphate (ATP) concentration, membrane potential, content of biomacromolecule, and cell morphology. Furthermore, the effect of CBCE on the counts of Salmonella Typhimurium and color of cooked chicken during storage was studied. The results showed that the ZI, MIC, and MBC of CBCE against Salmonella Typhimurium were 12.9 ± 0.53-13.6 ± 0.14 mm, 40, and 80 mg/mL, respectively. In the process of inhibiting Salmonella Typhimurium by CBCE, the reduction of intracellular ATP concentration, cell membrane depolarization, leakage of protein and nucleic acid, and destruction of cell morphology were observed. Moreover, after treatments with CBCE, the growth of Salmonella Typhimurium in cooked chicken was significantly inhibited (p < 0.05) compared with the control group. No significant differences (p > 0.05) in lightness (L*), redness (a*), and yellowness (b*) values of cooked chicken were found between untreated and treated samples, as well as the color of cooked chicken treated with CBCE did not change significantly (p > 0.05) during the six days of storage. Overall, our findings suggested that CBCE exhibited the antibacterial effect against Salmonella Typhimurium, and had the potential to be used as a natural food preservative for the control of Salmonella Typhimurium in chicken products.

Adenosine-Metabolizing Enzymes, Adenosine Kinase and Adenosine Deaminase, in Cancer.

The immunosuppressive effect of adenosine in the microenvironment of a tumor is well established. Presently, researchers are developing approaches in immune therapy that target inhibition of adenosine or its signaling such as CD39 or CD73 inhibiting antibodies or adenosine A2A receptor antagonists. However, numerous enzymatic pathways that control ATP-adenosine balance, as well as understudied intracellular adenosine regulation, can prevent successful immunotherapy. This review contains the latest data on two adenosine-lowering enzymes: adenosine kinase (ADK) and adenosine deaminase (ADA). ADK deletes adenosine by its phosphorylation into 5′-adenosine monophosphate. Recent studies have revealed an association between a long nuclear ADK isoform and an increase in global DNA methylation, which explains epigenetic receptor-independent role of adenosine. ADA regulates the level of adenosine by converting it to inosine. The changes in the activity of ADA are detected in patients with various cancer types. The article focuses on the biological significance of these enzymes and their roles in the development of cancer. Perspectives of future studies on these enzymes in therapy for cancer are discussed.

Dynamic Assembly of DNA Nanostructures in Living Cells for Mitochondrial Interference.

Constructing artificial dynamic architectures inside cells to rationally interfere with organelles is emerging as an efficient strategy to regulate the behaviors and fate of cells, thus providing new routes for therapeutics. Herein, we develop an intracellular K+-mediating dynamic assembly of DNA tetrahedrons inside cells, which realizes efficient mitochondrial interference and consequent regulation on the energy metabolism of living cells. In the designer DNA tetrahedron, one vertex was modified with triphenylphosphine (TPP) for mitochondrial targeting, and the other three vertexes were tethered with guanine-rich sequences that could realize K+-mediating formation of intermolecular G-quadruplexes, which consequently led to the assembly of DNA tetrahedrons to form aggregates in the cytoplasm. The DNA aggregates specially targeted mitochondria and served as a polyanionic barrier for substance communication, thus generating a significant inhibition effect on the aerobic respiration function of mitochondria and the associated glycolysis process, which consequently reduced the production of intracellular adenosine triphosphate (ATP). The lack of ATP impeded the formation of lamellipodium that was essential for the movement of cells, consequently resulting in a significant inhibitory effect on cell migration. Remarkably, the migration capacity was suppressed by as high as 50% for cancer cells. This work provides a new strategy for the manipulation of organelles via the endogenous molecule-mediating dynamic assembly of exogenous artificial architectures inside living cells, which is envisioned to have great potential in precise biomedicine.

Antibacterial mechanism of Protocatechuic acid against Yersinia enterocolitica and its application in pork

Protocatechuic acid (PCA) has adequate antibacterial activity against foodborne pathogens; however, its antibacterial mechanism has been rarely investigated. Therefore, this study attempts to elucidate the antibacterial mechanism of PCA against Yersinia enterocolitica . The corresponding results showed that PCA had a satisfactory antibacterial effect on Y. enterocolitica with a minimum inhibitory concentration (MIC) of 2.5 mg/mL. Accordingly, depolarization of the cell membrane, reduction of intracellular pH (pH in ) and adenosine triphosphate (ATP), leakage of K + and destruction of cell morphology were observed in this study, which indicated that PCA changed the membrane permeability of Y. enterocolitica . According to the metabolomics analysis, PCA induced the differential expression of the metabolites of Y. enterocolitica . In addition, enrichment analysis of metabolic pathways demonstrated that PCA disordered energy metabolism and amino acid biosynthesis of Y. enterocolitica . PCA also exhibited a notable inhibitory effect on Y. enterocolitica in pork. According to the corresponding findings, PCA may possess potential antibacterial properties in controlling Y. enterocolitica contamination within food industry. • Protocatechuic acid showed high antibacterial activity against Y. enterocolitica. • Protocatechuic acid destroyed the cell membrane structure of Y. enterocolitica. • Protocatechuic acid disordered energy metabolism and amino acid metabolism. • Protocatechuic acid extended the shelf life of chilled pork.

Transcriptomics analyses and biochemical characterization of Aspergillus flavus spores exposed to 1-nonanol.

The exploitation of plant volatile organic compounds as biofumigants to control postharvest decaying of agro-products has received considerable research attention. Our previous study reported that 1-nonanol, the main constituent of cereal volatiles, can inhibit Aspergillus flavus growth and has the potential as a biofumigant to control the fungal spoilage of cereal grains. However, the antifungal mechanism of 1-nonanol against A. flavus is still unclear at the molecular level. In this study, the minimum inhibitory concentration and minimum fungicidal concentration of 1-nonanol against A. flavus spores were 2 and 4 μL/mL, respectively. Scanning electron microscopy revealed that the 1-nonanol can distort the morphology of A. flavus spore. Annexin V-FITC/PI double staining showed that 1-nonanol induced phosphatidylserine eversion and increased membrane permeability of A. flavus spores. Transcriptional profile analysis showed that 1-nonanol treatment mainly affected the expression of genes related to membrane damage, oxidative phosphorylation, blockage of DNA replication, and autophagy in A. flavus spores. Flow cytometry analysis showed that 1-nonanol treatment caused hyperpolarization of mitochondrial membrane potential and accumulation of reactive oxygen species in A. flavus spores. 4',6-diamidino-2-phenylindole staining showed that treatment with 1-nonanol destroyed the DNA. Biochemical analysis results confirmed that 1-nonanol exerted destructive effects on A. flavus spores by decreasing intracellular adenosine triphosphate content, reducing mitochondrial ATPase activity, accumulating hydrogen peroxide and superoxide anions, and increasing catalase and superoxide dismutase enzyme activities. This study provides new insights into the antifungal mechanisms of 1-nonanol against A. flavus. KEY POINTS: • 1-Nonanol treatment resulted in abnormal morphology of A. flavus spores. • 1-Nonanol affects the expression of key growth-related genes of A. flavus. • The apoptosis of A. favus spores were induced after exposed to 1-nonanol.

ATP-responsive biomolecular condensates tune bacterial kinase signaling.

Biomolecular condensates formed via liquid-liquid phase separation enable spatial and temporal organization of enzyme activity. Phase separation in many eukaryotic condensates has been shown to be responsive to intracellular adenosine triphosphate (ATP) levels, although the consequences of these mechanisms for enzymes sequestered within the condensates are unknown. Here, we show that ATP depletion promotes phase separation in bacterial condensates composed of intrinsically disordered proteins. Enhanced phase separation promotes the sequestration and activity of a client kinase enabling robust signaling and maintenance of viability under the stress posed by nutrient scarcity. We propose that a diverse repertoire of condensates can serve as control knobs to tune enzyme sequestration and reactivity in response to the metabolic state of bacterial cells.

Kaempferol increases intracellular ATP content in C2C12 myotubes under hypoxic conditions by suppressing the HIF-1α stabilization and/or by enhancing the mitochondrial complex IV activity

Kaempferol (KMP) has numerous important biological functions, and we recently showed that it remarkably increased intracellular adenosine triphosphate (ATP) content in C2C12 myotubes under hypoxic conditions. Since intracellular ATP is generated by aerobic energy metabolism or anaerobic glycolysis, hypoxia inducible factor-1α (HIF-1α) has been shown to be associated with metabolic remodeling and causes metabolic shift from aerobic energy metabolism to anaerobic glycolysis in response to hypoxic conditions. Here, we investigate the effects of KMP under hypoxic conditions on the stabilization of HIF-1α in C2C12 myotubes and its underlying molecular mechanisms. Constitutive HIF-1α protein expression was observed in C2C12 myotubes, and its expression under hypoxic conditions was remarkably suppressed by KMP by reducing its stability; thus, resulting in an increase in ATP content. Furthermore, KMP strikingly increased the ubiquitination of HIF-1α and promoted its degradation via the ubiquitin proteasome system. Inhibition of HIF-1α by KMP resulted in the abrogation of the expression of glycolytic enzymes such as lactate dehydrogenase A and pyruvate dehydrogenase kinase isozyme 1. In addition, the metabolome profiling showed that KMP promoted oxidative energy production, while the mitochondrial complex activity assay indicated that KMP increased the activity of mitochondrial complex IV. Finally, we showed that KMP inhibited HIF-1α expression and increased intracellular ATP content in the soleus muscle of rats. Taken together, these results suggest that KMP increases intracellular ATP content under hypoxic conditions by suppressing the HIF-1α stabilization and/or by enhancing the mitochondrial complex IV activity in muscle.