Intracellular Adenosine Triphosphate Research Articles

Serine Improves Lactic Acid Stress Tolerance and Ethanol Production in Zygosaccharomyces bailii in Baijiu Fermentation.

Lactic acid is the primary inhibitor of the growth and ethanol production of yeasts in Baijiu fermentation. Certain amino acids have been found to be related to stress tolerance in yeasts. This study explored the effect of lactic acid stress on the ethanol-producing yeast Zygosaccharomyces bailii and evaluated the ability of serine to increase the lactic acid tolerance of Z. bailii in vitro. Serine significantly improved Z. bailii viability by 16.5% and ethanol production by 226.6% under lactic acid stress. Under lactic acid stress, serine supplementation led to an increase of 41.9% in cell wall integrity, 31.9% in cell membrane integrity, 296.6% in intracellular adenosine triphosphate (ATP), and 18.4% in the mitochondrial membrane potential. Finally, field emission scanning electron microscopy (FESEM) indicated that serine supplementation maintained the cell shape and reduced cell leakage. This study revealed a novel lactic acid tolerance mechanism of core functional yeasts during Jiang-flavor Baijiu fermentation.

Berberine derivative DCZ0358 induce oxidative damage by ROS-mediated JNK signaling in DLBCL cells

The most common neoplasm among adult lymphomas is diffuse large B-cell lymphoma (DLBCL), typically characterized by pain-free and progressive lymph node enlargement. Due to high heterogeneity of DLBCL, 30–40 % of patients are resistant to R-CHOP standard chemoimmunotherapy. DCZ0358 is a new compound designed and synthesized from berberine by our group and the molecular mechanism by which it inhibited DLBCL growth has attracted our widespread attention. In this study, we employed the CCK8 assay to reveal that DCZ0358 inhibited proliferation in a dependent manner of time and dosage of DLBCL cells. Moreover, flowcytometry and western blot results showed that DCZ0358 downregulated the expression of CDK4, CDK6 and CyclinD1 to block cell cycle progression in G0/G1 phase. Furthermore, DCZ0358 enhanced mitochondrial membrane potential depolarization, promoted mitochondrial permeability transport pore openness, increased cytoplastic Ca2+ levels and decreased intracellular adenosine triphosphate production, which led to mitochondrial dysfunction. In particular, DCZ0358 treatment triggered cell apoptosis and elevated intracellular reactive oxygen species (ROS) levels, which subsequently mediated JNK pathway activation. Further research indicated the pre-treatment with ROS scavenger N-acetylcysteine (NAC) and JNK inhibitor SP600125 could partially attenuate apoptosis and DNA damage triggered by DCZ0358. Most importantly, DCZ0358 exhibited synergistic anti-tumor effects when combined with etoposide, a common clinical anti-DLBCL drug, both in vitro and certainly in vivo. Above results demonstrated anti-tumor molecular mechanism of DCZ0358 in DLBCL cells and highlighted the ROS/JNK/DNA damage pathway as a potential target in therapies, which have implications for the development of more effective clinical treatments for DLBCL.

Microbial thermogenesis is dependent on ATP concentrations and the protein kinases ArcB, GlnL, and YccC.

Organisms necessarily release heat energy in their pursuit of survival. This process is known as cellular thermogenesis and is implicated in many processes from cancer metabolism to spontaneous farm fires. However, the molecular basis for this fundamental phenomenon is yet to be elucidated. Here, we show that the major players involved in the cellular thermogenesis of Escherichia coli are the protein kinases ArcB, GlnL, and YccC. We also reveal the substrate-level control of adenosine triphosphate (ATP)-driven autophosphorylation that governs cellular thermogenesis. Specifically, through live cell microcalorimetry, we find these regulatory proteins, when knocked out in a model E. coli strain, dysregulate cellular thermogenesis. This dysregulation can be seen in an average 25% or greater increase in heat output by these cells. We also discover that both heat output and intracellular ATP levels are maximal during the late log phase of growth. Additionally, we show that microbial thermogenesis can be engineered through overexpressing glnL. Our results demonstrate a correlation between ATP concentrations in the cell and a cell's ability to generate excess heat. We expect this work to be the foundation for engineering thermogenically tuned organisms for a variety of applications.

A spatially-controlled DNA triangular prism nanomachine for AND-gated intracellular imaging of ATP in acidic microenvironment.

ADNA triangular prism nanomachine (TPN)-based logic device for intracellular AND-gated imaging of adenosine triphosphate (ATP)has been constructed. By using i-motif sequences and ATP-binding aptamers as logic control units, the TPN logic device is qualified to respond to the acidic environment and ATP in cancer cell lysosomes. Once internalized into the lysosome, the specific acidic microenvironment in lysosome causes the i-motif sequence to fold into a tetramer, resulting in compression of DNA tri-prism. Subsequently, the split ATP aptamer located at the tip of the collapsed triangular prism binds stably to ATP, which results in the fluorescent dyes (Cy3 and Cy5) modified at the ends of the split aptamer being in close proximity to each other, allowing Förster Resonance Energy Transfer (FRET) to occur. The FRET signals are excited at a wavelength of 543 nm and can be collected within the emission range of 646-730 nm. This enables the precise imaging of ATP within a cell. We also dynamically operate AND logic gates in living cells by modulating intracellular pH and ATP levels with the help of external drugs. Owing to the AND logic unit on TPN itcan simultaneously recognize two targets and give corresponding intelligent logic judgment via imaging signal output. Theaccuracy of molecular diagnosis of cancer can be improved thuseliminating the false positive signal of single target-based detection. Hence, this space-controlled TPN-based logical sensing platform greatly avoids sensitivity to extracellular targets during the cell entry process, providing a useful tool for high-precision imaging of the cancer cell's endogenous target ATP.

Synergistic bactericidal mechanisms of RF energy simultaneously combined with cinnamon essential oil or epsilon-polylysine against Salmonella revealed at cellular and metabolic levels

Radio frequency (RF) heating and antimicrobials are considered to be effective methods for inactivating food pathogens. This study explored the bactericidal effects against Salmonella of RF heating combined with two kinds of natural antimicrobials possessing different hydrophobic properties and their synergistic bactericidal mechanisms. Results showed that RF heating caused sublethal damage to bacterial cells and enhanced the interaction of cells and antimicrobials, leading to synergistic bactericidal effects of the simultaneous combination of RF heating and antimicrobials. The combination of RF heating and ε-polylysine (ε-PL) further promoted cell morphological alteration, raised membrane permeability, intracellular adenosine triphosphate (ATP) leakage and intracellular reactive oxygen species (ROS) accumulation compared to individual treatment. The simultaneous combination of RF heating and cinnamon essential oil nanoemulsion (CEON) also further enhanced membrane permeability and ROS accumulation compared to individual treatment, but impacts were less than those in the combination of RF heating and ε-PL. The major synergistic bactericidal mechanism of RF heating and CEON was significantly inhibiting intracellular ATP synthesis. The untargeted metabolomics analysis revealed that the combined treatments enhanced disturbances to multiple intracellular metabolisms compared to individual treatment, thus leading to synergistic bactericidal effects against Salmonella. These results provide an in-depth understanding of the synergistic bactericidal mechanisms of the combination of RF heating and natural antimicrobials from cellular and metabolic levels.

Reduced lysosomal activity and increased amyloid beta accumulation in silica-coated magnetic nanoparticles-treated microglia.

Nanoparticles have been used in neurological research in recent years because of their blood-brain barrier penetration activity. However, their potential neuronanotoxicity remains a concern. In particular, microglia, which are resident phagocytic cells, are mainly exposed to nanoparticles in the brain. We investigated the changes in lysosomal function in silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)]-treated BV2 murine microglial cells. In addition, we analyzed amyloid beta (Aβ) accumulation and molecular changes through the integration of transcriptomics, proteomics, and metabolomics (triple-omics) analyses. Aβ accumulation significantly increased in the 0.1μg/μl MNPs@SiO2(RITC)-treated BV2 cells compared to the untreated control and 0.01μg/μl MNPs@SiO2(RITC)-treated BV2 cells. Moreover, the MNPs@SiO2(RITC)-treated BV2 cells showed lysosomal swelling, a dose-dependent reduction in proteolytic activity, and an increase in lysosomal swelling- and autophagy-related protein levels. Moreover, proteasome activity decreased in the MNPs@SiO2(RITC)-treated BV2 cells, followed by a concomitant reduction in intracellular adenosine triphosphate (ATP). By employing triple-omics and a machine learning algorithm, we generated an integrated single molecular network including reactive oxygen species (ROS), autophagy, lysosomal storage disease, and amyloidosis. In silico analysis of the single triple omics network predicted an increase in ROS, suppression of autophagy, and aggravation of lysosomal storage disease and amyloidosis in the MNPs@SiO2(RITC)-treated BV2 cells. Aβ accumulation and lysosomal swelling in the cells were alleviated by co-treatment with glutathione (GSH) and citrate. These findings suggest that MNPs@SiO2(RITC)-induced reduction in lysosomal activity and proteasomes can be recovered by GSH and citrate treatment. These results also highlight the relationship between nanotoxicity and Aβ accumulation.

Aminooxyacetic acid hemihydrochloride leads to decreased intracellular ATP levels and altered cell cycle of prostate cancer cells by suppressing energy metabolism

The second most common cancer among men is prostate cancer, which is also the fifth leading reason for male cancer deaths worldwide. Bone metastases are the main factor affecting the prognosis of prostate cancer. Consequently, antitumor and anti-prostate cancer-induced bone destruction medicines are urgently needed. We previously discovered that aminooxyacetic acid hemihydrochloride (AOAA) suppressed bone resorption and osteoclast growth by decreasing adenosine triphosphate (ATP) production and limiting oxidative phosphorylation (OXPHOS). Here, we evaluated the impacts of AOAA on prostate cancer RM-1 cells in vitro. It’s found that AOAA significantly inhibited cell proliferation, migration, and invasiveness, decreased ATP levels, increased ROS, halted the cell cycle phase, and triggered apoptosis. AOAA also decreased mitochondrial membrane potential and the ability to uptake glucose, suggesting that the antitumor effects of AOAA were expressed through the inhibition of OXPHOS and glycolysis. Furthermore, we assessed the effects of AOAA in vivo using a prostate cancer-induced bone osteolysis mice model. AOAA also delayed tumor growth and bone destruction in vivo. On the whole, our findings imply that AOAA may potentially have therapeutic effects on prostate cancer and prostate cancer-induced osteolysis.

CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.

EGR1-mediated metabolic reprogramming to oxidative phosphorylation contributes to ibrutinib resistance in B-cell lymphoma

AbstractThe use of Bruton tyrosine kinase inhibitors, such as ibrutinib, to block B-cell receptor signaling has achieved a remarkable clinical response in several B-cell malignancies, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Acquired drug resistance, however, is significant and affects the long-term survival of these patients. Here, we demonstrate that the transcription factor early growth response gene 1 (EGR1) is involved in ibrutinib resistance. We found that EGR1 expression is elevated in ibrutinib-resistant activated B-cell–like subtype DLBCL and MCL cells and can be further upregulated upon ibrutinib treatment. Genetic and pharmacological analyses revealed that overexpressed EGR1 mediates ibrutinib resistance. Mechanistically, TCF4 and EGR1 self-regulation induce EGR1 overexpression that mediates metabolic reprogramming to oxidative phosphorylation (OXPHOS) through the transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation increases intracellular adenosine triphosphate production, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with metformin or IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting EGR1-mediated metabolic reprogramming to OXPHOS with metformin or IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

In-vitro antibacterial activity and mechanism of Monarda didyma essential oils against Carbapenem-resistant Klebsiella pneumoniae

To fight the global epidemic of drug-resistant bacteria, essential oils have gained increasing attention as a new source of antibiotics. The antimicrobial activity of Monarda didyma essential oils (MDEO) for the Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were determined by agar disc diffusion assay and broth microdilution assay. To further understand MDEO efficacy, a time-growth curve was performed. The biofilm formation of CRKP were determined by crystalline violet staining method, additionally, changes in intracellular Adenosine triphosphate (ATP), protein, Alkaline phosphatase (AKP) activities, and membrane integrity were investigated to assess the influence of MDEO on cell membrane damage. Finally, the activities of key enzymes in the tricarboxylic acid (TCA) pathways and pentose phosphate (PPP) pathways were examined to determine the effect of MDEO on the respiratory metabolism of CRKP. This study presents the antibacterial mechanism of MDEO against CRKP with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 1.25 mg/ml. To understand MDEO efficacy, a time-kill kinetics approach was performed. The bactericidal effect of MDEO was evident at 2 h compared to the control at its MIC and 2MIC. Surface electron microscopic and ATP assay studies provided evidence for the multi-target action of MDEO against CRKP. MDEO could inhibit CRKP biofilm formation. MDEO could also cause irreversible damage to the CRKP cell membrane, resulting in the leakage of biological macromolecules (protein, ATP) and the reduction of intracellular enzymes (AKP) activities. Finally, MDEO affected the pathways of respiratory metabolism, such as PPP and TCA pathways. MDEO could reduce the activity of key enzymes (Glucose-6-phosphate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) in the PPP and TCA pathways to exert its biological effects against CRKP. These results suggest MDEO can exert inhibitory effects on CRKP, and potential mechanisms of action including inhibition of biofilm formation, damage of cell membrane structure and inhibition of energy metabolism.

Black Phosphorus/MnO2 Nanocomposite Disrupting Bacterial Thermotolerance for Efficient Mild-Temperature Photothermal Therapy.

The emergence of multi-drug resistant (MDR) pathogens is a major public health concern, posing a substantial global economic burden. Photothermal therapy (PTT) at mild temperature presents a promising alternative to traditional antibiotics due to its biological safety and ability to circumvent drug resistance. However, the efficacy of mild PTT is limited by bacterial thermotolerance. Herein, a nanocomposite, BP@Mn-NC, comprising black phosphorus nanosheets and a manganese-based nanozyme (Mn-NZ) is developed, which possesses both photothermal and catalytic properties. Mn-NZ imparts glucose oxidase- and peroxidase-like properties to BP@Mn-NC, generating reactive oxygen species (ROS) that induce lipid peroxidation and malondialdehyde accumulation across the bacterial cell membrane. This process disrupts unprotected respiratory chain complexes exposed on the bacterial cell membrane, leading to a reduction in the intracellular adenosine triphosphate (ATP) content. Consequently, mild PTT mediated by BP@Mn-NC effectively eliminates MDR infections by specifically impairing bacterial thermotolerance because of the dependence of bacterial heat shock proteins (HSPs) on ATP molecules for their proper functioning. This study paves the way for the development of a novel photothermal strategy to eradicate MDR pathogens, which targets bacterial HSPs through ROS-mediated inhibition of bacterial respiratory chain activity.

Dual Effects of Feed-Additive-Derived Chelerythrine in Combating Mobile Colistin Resistance

The emergence and spread of the mobile colistin-resistance gene, mcr-1, and its variants pose a challenge to the use of colistin, a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant (XDR) Gram-negative pathogens. Antibiotic adjuvants are a promising strategy to enhance the efficacy of colistin against colistin-resistant pathogens; however, few studies have considered the effects of adjuvants on limiting resistance-gene transmission. We found that chelerythrine (4 mg∙L−1) derived from Macleaya cordata extract, which is used as an animal feed additive, reduced the minimal inhibitory concentration (MIC) of colistin against an mcr-1 positive Escherichia coli (E. coli) strain by 16-fold (from 2.000 to 0.125 mg∙L−1), eliminated approximately 104 colony-forming units (CFUs) of an mcr-1-carrying strain in a murine intestinal infection model, and inhibited the conjugation of an mcr-1-bearing plasmid in vitro (by > 100-fold) and in a mouse model (by up to 5-fold). A detailed analysis revealed that chelerythrine binds to phospholipids on bacterial membranes and increases cytoplasmic membrane fluidity, thereby impairing respiration, disrupting proton motive force (PMF), generating reactive oxygen species (ROS), and decreasing intracellular adenosine triphosphate (ATP) levels, which subsequently downregulates mcr-1 and conjugation-associated genes. These dual effects of chelerythrine can expand the use of antibiotic adjuvants and may provide a new strategy for circumventing mobile colistin resistance.

Влияние внутриклеточной регуляции метаболизма на популяционный состав лимфоцитов периферической крови человека

Metabolic activity has a significant impact on the differentiation, proliferation and functioning of T cells. Different lymphocyte subpopulations use, to varying degrees, glycolysis and mitochondrial metabolism, whose main regulators are hypoxia-inducible factor 1-alpha (HIF-1α) and sirtuin 3 (SIRT3), respectively. The purpose of this paper was to study changes in the population composition of peripheral blood lymphocytes in humans depending on the level of the intracellular metabolic regulators SIRT3 and HIF-1α. Materials and methods. 227 residents of the city of Arkhangelsk and the Arkhangelsk Region were examined (mean age 42 ± 11 years). Absolute lymphocyte count was determined using the Sysmex XS 500i haematology analyser, while CD3+, CD4+, CD8+, CD10+, CD25+ and CD95+ phenotypes content, by indirect immunoperoxidase reaction. Intracellular adenosine triphosphate (ATP) concentration was measured using the luciferase bioluminescence method. HIF-1α and SIRT3 concentrations were measured in lymphocyte lysate using enzyme immunoassay. To divide the total sample into groups according to SIRT3 and HIF-1α content, k-means clustering was utilized. Results. Changes in SIRT3 and HIF-1α intracellular concentrations correlated with ATP concentration. It was found that in the group with high HIF-1α content, the proportion of CD4+, CD8+, CD10+ and CD25+ lymphocytes was greater than in the group with high SIRT3 concentration, which had a greater proportion of CD95+ lymphocytes. Thus, the content of intracellular metabolic regulators that regulate ATP production pathways in the cell, i.e. oxidative phosphorylation (SIRT3) and glycolysis (HIF-1α), affects the population composition of lymphocytes and is therefore important for assessing the immune response.

Unveiling the Crucial Roles of O2•- and ATP in Hepatic Ischemia-Reperfusion Injury Using Dual-Color/Reversible Fluorescence Imaging.

Hepatic ischemia-reperfusion injury (HIRI) is mainly responsible for morbidity or death due to graft rejection after liver transplantation. During HIRI, superoxide anion (O2•-) and adenosine-5'-triphosphate (ATP) have been identified as pivotal biomarkers associated with oxidative stress and energy metabolism, respectively. However, how the temporal and spatial fluctuations of O2•- and ATP coordinate changes in HIRI and particularly how they synergistically regulate each other in the pathological mechanism of HIRI remains unclear. Herein, we rationally designed and successfully synthesized a dual-color and dual-reversible molecular fluorescent probe (UDP) for dynamic and simultaneous visualization of O2•- and ATP in real-time, and uncovered their interrelationship and synergy in HIRI. UDP featured excellent sensitivity, selectivity, and reversibility in response to O2•- and ATP, which rendered UDP suitable for detecting O2•- and ATP and generating independent responses in the blue and red fluorescence channels without spectral crosstalk. Notably, in situ imaging with UDP revealed for the first time synchronous O2•- bursts and ATP depletion in hepatocytes and mouse livers during the process of HIRI. Surprisingly, a slight increase in ATP was observed during reperfusion. More importantly, intracellular O2•-─succinate dehydrogenase (SDH)─mitochondrial (Mito) reduced nicotinamide adenine dinucleotide (NADH)─Mito ATP─intracellular ATP cascade signaling pathway in the HIRI process was unveiled which illustrated the correlation between O2•- and ATP for the first time. This research confirms the potential of UDP for the dynamic monitoring of HIRI and provides a clear illustration of HIRI pathogenesis.

NIR‐II Photothermal Activation of TRPV1 Channels for Intracellular Magnesium Regulation by Porous Pd@Pt Core–Shell Nanostructure to Reverse Tumor Multidrug Resistance

AbstractMultidrug resistance (MDR) caused by overexpressed P‐glycoprotein (P‐gp) in cell membrane is the main barrier for clinical tumor chemotherapy. P‐gp can pump the chemical drugs out of tumor cells depending on ATP‐provided energy. Herein, a photothermal‐driven intracellular magnesium ion (Mg2+) regulation strategy is proposed to reverse drug resistance through constructing Mg2+‐ and doxorubicin (DOX, as model drug)‐loaded bimetallic Pd@Pt nanostructure (DPd@PtM). Although DPd@PtM can deliver Mg2+ into tumor cells through endocytosis, large amount of Mg2+ releases outside cells. To this end, the photothermal effect of Pd@Pt nanostructure in the second near‐infrared region is expected to activate the thermosensitive transient receptor potential cation channel subfamily V member 1 (TRPV1) channel for extracellular released Mg2+ influx. Intercellular Mg2+ accumulation suppresses tricarboxylic acid cycle to block intracellular adenosine triphosphate (ATP) production (cutoff energy supply for P‐gp) and reduce O2 consumption (downregulate P‐gp expression), then inhibiting P‐gp‐mediated tumor MDR. Both in vitro and in vivo results demonstrate that DPd@PtM can open TRPV1 channel to elevate Mg2+ level and then inhibit the P‐gp activity to enhance intracellular DOX concentration for chemotherapy. It is believed that this photothermal‐mediated tumor Mg2+ regulation therapy based on reversing MDR is a promising strategy to kill cancer cells.

Antibacterial mechanism of vanillin against Escherichia coli O157: H7

Vanillin, a plant-derived antimicrobial volatile substance, has potential microbial control applications in the food industry. However, the effect of vanillin on the food-borne pathogen Escherichia coli (E. coli) O157:H7 has not been well studied. This study aims to explore the antibacterial mechanism of vanillin against E. coli O157:H7. The minimum inhibitory concentration (MIC) and antibacterial effect of vanillin were determined by microdilution. Scanning electron microscopy (SEM) was used to observe the damage of vanillin to the cell membrane, while cell membrane potential and the leakage of nucleic acid protein were measured to explore the effect of vanillin on the membrane system. Confocal laser scanning and intracellular adenosine triphosphate (ATP) concentration determination were utilized to investigate the effects of vanillin on the energy, life, and death of E. coli. Finally, transcriptome sequencing was conducted to investigate the gene expression differences induced by vanillin treatment. The results showed that vanillin treatment effectively controlled E. coli O157:H7 with an MIC of 2 mg/mL. After treatment, damage to the membrane system, depolarization of the membrane, and leakage of nucleic acid and protein were observed. Meanwhile, vanillin treatment caused decreased ATP content and cell death. Transcriptome analysis showed that vanillin treatment significantly affected the expression of genes involved in cell membrane formation, tricarboxylic acid (TCA) cycling pathway, and oxidative phosphorylation pathway in E. coli O157:H7. In conclusion, membrane damage and energy metabolism disruption are important mechanisms of vanillin's inhibitory effect on E. coli O157:H7. This study provides new insights into the molecular reaction mechanism of vanillin against E. coli O157:H7, highlighting its potential as an antibacterial substance for preventing E. coli contamination in the food industry.

A Novel NCI-H69AR Drug-Resistant Small-Cell Lung Cancer Mini-Tumor Model for Anti-Cancer Treatment Screening.

Small-cell lung cancer is a fast-growing carcinoma with a poor prognosis and a high level of relapse due to multi-drug resistance (MDR). Genetic mutations that lead to the overexpression of efflux transporter proteins can contribute to MDR. In vitro cancer models play a tremendous role in chemotherapy development and the screening of possible anti-cancer molecules. Low-cost and simple in vitro models are normally used. Traditional two-dimensional (2D) models have numerous shortcomings when considering the physiological resemblance of an in vivo setting. Three-dimensional (3D) models aim to bridge the gap between conventional 2D models and the in vivo setting. Some of the advantages of functional 3D spheroids include better representation of the in vivo physiology and tumor characteristics when compared to traditional 2D cultures. During this study, an NCI-H69AR drug-resistant mini-tumor model (MRP1 hyperexpressive) was developed by making use of a rotating clinostat bioreactor system (ClinoStar®; CelVivo ApS, Odense, Denmark). Spheroid growth and viability were assessed over a 25-day period to determine the ideal experimental period with mature and metabolically stable constructs. The applicability of this model for anti-cancer research was evaluated through treatment with irinotecan, paclitaxel and cisplatin for 96 h, followed by a 96 h recovery period. Parameters measured included planar surface area measurements, estimated glucose consumption, soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase levels, histology and efflux transporter gene expression. The established functional spheroid model proved viable and stable during the treatment period, with retained relative hyperexpression of the MRP1 efflux transporter gene but increased expression of the P-gp transporter gene compared to the cells cultured in 2D. As expected, treatment with the abovementioned anti-cancer drugs at clinical doses (100 mg/m2 irinotecan, 80 mg/m2 paclitaxel and 75 mg/m2 cisplatin) had minimal impact on the drug-resistant mini-tumors, and the functional spheroid models were able to recover following the removal of treatment.

The role of pyruvate-induced enhancement of oxygen metabolism in extracellular purinergic signaling in the post-cardiac arrest rat model.

Purine nucleotide adenosine triphosphate (ATP) is a source of intracellular energy maintained by mitochondrial oxidative phosphorylation. However, when released from ischemic cells into the extracellular space, they act as death-signaling molecules (eATP). Despite there being potential benefit in using pyruvate to enhance mitochondria by inducing a highly oxidative metabolic state, its association with eATP levels is still poorly understood. Therefore, while we hypothesized that pyruvate could beneficially increase intracellular ATP with the enhancement of mitochondrial function after cardiac arrest (CA), our main focus was whether a proportion of the raised intracellular ATP would detrimentally leak out into the extracellular space. As indicated by the increased levels in systemic oxygen consumption, intravenous administrations of bolus (500 mg/kg) and continuous infusion (1000 mg/kg/h) of pyruvate successfully increased oxygen metabolism in post 10-min CA rats. Plasma ATP levels increased significantly from 67 ± 11 nM before CA to 227 ± 103 nM 2 h after the resuscitation; however, pyruvate administration did not affect post-CA ATP levels. Notably, pyruvate improved post-CA cardiac contraction and acidemia (low pH). We also found that pyruvate increased systemic CO2 production post-CA. These data support that pyruvate has therapeutic potential for improving CA outcomes by enhancing oxygen and energy metabolism in the brain and heart and attenuating intracellular hydrogen ion disorders, but does not exacerbate the death-signaling of eATP in the blood.

Real-time monitoring ATP variation in human cancer organoids for a long term by DNA-based nanosensor

Cancer organoids have become promising tools for predicting drug responses on many different types of cancer. Detecting the adenosine triphosphate (ATP) has currently been considered as a decisive test to profile the growth status and drug responses of organoids. ATP profiling using commercial ATP detection kits, which involve cell lysis, can be performed at a single time spot, causing a clinical dilemma of selecting the optimal time spot to adopt diverse cancer types and patients. This study provides a feasible solution to this dilemma by developing a DNA-based ATP nanosensor to realize real-time ATP monitoring in organoids for a long term. The employment of DNA materials ensures high biocompatibility and low cytotoxicity, which are crucial for fragile organoids; The usage of tetrahedral DNA framework ensures cell permeability and intracellular ATP detection; The introduction of ATP-mediated molecular replacement ensures the high sensitivity and selectivity of ATP recognition. These features result in the first successful attempt on real-time monitoring ATP in organoids for up to 26 days and gaining growth status curves for the whole duration of a drug sensitivity test on human lung cancer organoids.

Highly sensitive and stable fluorescent aptasensor based on an exonuclease III-assisted amplification strategy for ATP detection

Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L−1 to μmol L−1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.