CRISPR-Cas12a-mediated dual-enzyme cascade amplification for sensitive colorimetric detection of HPV-16 target and ATP.

The establishment of sensitive and facile colorimetric platform based on the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is of great significance for in vitro diagnosis. Herein, we develop a dual-enzyme cascade amplification strategy based on CRISPR-Cas12a and glucose oxidase (GOx) for instrument-free and sensitive detection of target analytes. HPV-16 DNA as the model nucleic acid target directly initiated CRISPR-Cas12a-based signal transduction, resulting in the enzymatic cleavage of ssDNA linker and the release of GOx from magnetic nanoparticles 1 (MNPs1). Following simple magnetic separation, the supernatant containing GOx was taken out and used to catalyze the substrate, resulting in a visually detectable color change. The detection limit (LOD) of HPV-16 DNA was as low as 1 pM, and the entire process could be completed within 70 min without the need for expensive equipment. Notably, the dual-enzyme cascade amplification strategy was successfully applied to the detection of non-nucleic acid targets, such as ATP, via a simple signal transduction process. The visual LOD for ATP detection reaches 2.5 µM. The approach provides a robust, sensitive and reliable point-of-care biosensing platform for the detection of target analytes.

Fuzheng Huayu recipe alleviates liver fibrosis via inhibiting NLRP3 inflammasome activation in macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Fuzheng Huayu recipe (FZHY) is a commonly used Traditional Chinese Medicine formula for treating liver fibrosis in clinical settings. Despite its widespread use, the specific curative effects and underlying pharmacological mechanisms of FZHY in treating liver fibrosis are not yet fully understood. AIM AND STUDY: This study aims to investigate the antifibrotic mechanism of FZHY treatment by exploring its effects on the activation of NOD-like receptor protein 3 (NLRP3) inflammasome in macrophages. MATERIALS AND METHODS: In order to investigate the impact of FZHY on the activation and priming of NLRP3 inflammasome in clinical trials and animal experiments using immunohistochemistry and Western blotting. Twenty-four C57BL/6 mice were used to induce liver fibrosis by feeding a diet that contained 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). To study inflammasome function, Lipopolysaccharide (LPS)/adenine triphosphate (ATP) induced NLRP3 inflammasome activation was induced in bone marrow-derived macrophages (BMDMs) isolated from wild mice. The effects of macrophage NLRP3 inflammasome activation on the function of hepatic stellate cells (HSCs) were explored by treating primary HSCs with preconditioned media from BMDMs culture. RESULTS: FZHY treatment resulted in the downregulation of NLRP3 protein expression and inhibition of its priming and activation in both human fibrotic livers and DDC-induced liver fibrosis. Furthermore, FZHY was observed to block the activation of the NLRP3 inflammasome pathway, which can lead to excessive inflammatory cytokine release in supernatants and cell lysates in response to LPS and ATP. Lastly, treatment with FZHY was able to inhibit the activation of HSCs induced by supernatants from macrophages. CONCLUSIONS: FZHY has been shown to potentially prevent NLRP3 inflammasome activation in macrophages which can result in the suppression of HSCs activation. Ultimately, these effects may lead to the improvement of liver fibrosis. The ability of FZHY to act on this novel mechanism represents an important aspect of its therapeutic potential for liver fibrosis.

Network pharmacology and experimental validation on yangjing zhongyu decoction against diminished ovarian reserve.

ETHNOPHARMACOLOGICAL RELEVANCE: Diminished ovarian reserve (DOR) was considered a refractory reproductive endocrine condition that negatively affected female reproductivity. Yangjing Zhongyu Decoction (YJZYD) had effects on treating infertility. However, there were few studies on the mechanisms of YJZYD preserving ovarian reserve. AIM OF THE STUDY: To explore the possible mechanisms of YJZYD against DOR by UPLC-ESI-MS/MS, network pharmacology, and experimental validation. METHODS: The chemicals of YJZYD were measured by UPLC-ESI-MS/MS. The correlating targets of YJZYD and DOR were identified by the ETCM database, GeneCards database, and PubMed database. The common targets were employed with the DAVID database and visualized with the PPI network. GO and KEGG enrichment analyses were carried out to explore biological progression and pathways. In vivo experiments, energy production was assessed by ATP, and apoptosis rate was analyzed by TUNEL. The serum FSH, AMH, and E2 levels were evaluated by ELISA. Western blotting and immunohistochemistry were used to measure the expression of SIRT1, PGC1α, NRF1, COX IV, FSHR, CYP19A1, PI3K, p-Akt, Akt, Bcl-2, and Bax. RESULTS: 132 components in YJZYD were identified by UPLC-ESI-MS/MS. 149 overlapped targets were extracted from YJZYD and DOR, and the top 20 common targets included AKT1 and CYP19A1. ATP binding was involved in GO analysis. In the KEGG enrichment analysis, the metabolic pathway was the top, and the PI3K-Akt signaling pathway was included. In vivo experiments, YJZYD improved ovarian index and histomorphology. After YJZYD treatment, serum FSH, E2, and AMH were well-modulated, and the content of ATP was up-regulated. Besides, the expression of Bax was suppressed in ovarian tissue, while the expressions of SIRT1, PGC1α, NRF1, COX IV, FSHR, CYP19A1, PI3K, Bcl-2, and p-Akt/Akt were enhanced. CONCLUSION: YJZYD could attenuate reproductive endocrine disturbance and ovarian lesions in vivo by mediating steroidogenesis, energy metabolism, and cell apoptosis. This study uncovered the mechanisms of YJZYD against DOR, providing a theoretical basis for further study.

Quantifying ATP-Independent Nucleosome Chaperone Activity with Single-Molecule Methods.

The dynamics of histone-DNA interactions govern chromosome organization and regulates the processes of transcription, replication, and repair. Accurate measurements of the energies and the kinetics of DNA binding to component histones of the nucleosome under a variety of conditions are essential to understand these processes at the molecular level. To accomplish this, we employ three specific single-molecule techniques: force disruption (FD) with optical tweezers, confocal imaging (CI) in a combined fluorescence plus optical trap, and survival probability (SP) measurements of disrupted and reformed nucleosomes. Short arrays of positioned nucleosomes serve as a template for study, facilitating rapid quantification of kinetic parameters. These arrays are then exposed to FACT (FAcilitates Chromatin Transcription), a non-ATP-driven heterodimeric nuclear chaperone known to both disrupt and tether histones during transcription. FACT binding drives off the outer wrap of DNA and destabilizes the histone-DNA interactions of the inner wrap as well. This reorganization is driven by two key domains with distinct function. FD experiments show the SPT16 MD domain stabilizes DNA-histone contacts, while the HMGB box of SSRP1 binds DNA, destabilizing the nucleosome. Surprisingly, CI experiments do not show tethering of disrupted histones, but increased rates of histone release from the DNA. SI experiments resolve this, showing that the two active domains of FACT combine to chaperone nucleosome reassembly after the timely release of force. These combinations of single-molecule approaches show FACT is a true nucleosome catalyst, lowering the barrier to both disruption and reformation.

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.

Is the mitochondrial ATP synthesis solely H+-driven, K+,H+ symport-driven or K+/H+ antiport-driven?

Some recent literature experimental data indicate that the mitochondrial ATP synthesis might be not solely H+-driven, but K+,H+ symport-driven membrane potential-dependent, considered as a further development of Mitchell's chemiosmotic theory, in contrast to the anti-Mitchell's hypothesis of K+/H+ antiport-driven mechanism. In this short communication, the attention was pointed to a possible influence of the ionic strength of the used KCl incubation medium, versus of only K+ ions, and of the Mg2+-induced mitochondrial aggregation in the sucrose medium on the reported rates of the mitochondrial respiration and ATP synthesis. These observations were based on the own author's experimental works published earlier.

Cristae dynamics is modulated in bioenergetically compromised mitochondria.

Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here, we investigated whether and how cristae morphology and dynamics are dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential (ΔΨm), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A, and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not impair cristae dynamics. CCCP treatment leading to ΔΨm abrogation even enhanced cristae dynamics showing its ΔΨm-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange, and cristae membrane dynamics.

The pro-oncogenic protein IF1 does not contribute to the Warburg effect and is not regulated by PKA in cancer cells.

The endogenous inhibitor of mitochondrial F1Fo-ATPase (ATP synthase), IF1, has been shown to exert pro-oncogenic actions, including reprogramming of cellular energy metabolism (Warburg effect). The latter action of IF1 has been reported to be hampered by its PKA-dependent phosphorylation, but both reprogramming of metabolism and PKA-dependent phosphorylation are intensely debated. To clarify these critical issues, we prepared stably IF1-silenced clones and compared their bioenergetics with that of the three parental IF1-expressing cancer cell lines. All functional parameters: respiration rate, ATP synthesis rate (OXPHOS), and mitochondrial membrane potential were similar in IF1-silenced and control cells, clearly indicating that IF1 cannot inhibit the ATP synthase in cancer cells when the enzyme works physiologically. Furthermore, all cell types exposed to PKA modulators and energized with NAD+-dependent substrates or succinate showed similar OXPHOS rate regardless of the presence or absence of IF1. Therefore, our results rule out that IF1 action is modulated by its PKA-dependent phosphorylated/dephosphorylated state. Notably, cells exposed to a negative PKA modulator and energized with NAD+-dependent substrates showed a significant decrease of the OXPHOS rate matching previously reported inactivation of complex I. Overall, this study definitively demonstrates that IF1 inhibits neither mitochondrial ATP synthase nor OXPHOS in normoxic cancer cells and does not contribute to the Warburg effect. Thus, currently the protection of cancer cells from severe hypoxia/anoxia and apoptosis remain the only unquestionable actions of IF1 as pro-oncogenic factor that may be exploited to develop therapeutic approaches.

Selective turn-on sensing of adenosine diphosphate and phosphate anions by ruthenium (II) polypyridine anchored p-tert-butylcalix[4]arene platform.

BACKGROUND: Nucleoside polyphosphate (NPP) anions are important for enzymatic activity and should be monitored by scientists in industry and medicine. By elucidating enzyme kinetics and processes, it aids in the discovery of effective inhibitors and activators. Nucleoside polyphosphate (NPP) anions are used by kinases, GTPases, and glycosyltransferases (GTs). Phosphorylation of certain amino acid residues (Ser, Thr, and Tyr) on proteins requires the breakdown of ATP by protein kinases, which produces ADP. Protein kinases, breakdown of ATP, and NPP are the focus of oncology drug development because the aberrant control of kinase activity is a common cause of cancer. RESULTS: However, a discriminative turn-on fluorescent property is exhibited by non-fluorescent p-tertbutylcalix[4]arene modified 1,2,3-triazole containing bis-ruthenium polypyridyl complex (RL) upon the addition of phosphate anions such as (dihydrogen pyrophosphate (H2P2O72-) and dihydrogen phosphate (H2PO4-)) in CH3CN solvent and Adenosine Diphosphate (ADP) in CH3CN/HEPES (pH = 7.4) buffer (9/1, v/v). The probe RL shows a better-recognizing ability with pyrophosphate anion (H2P2O72-) than dihydrogen phosphate anion (H2PO4-). With H2P2O72- and H2PO4- anions, the RL detection limit was calculated to be as low as 83 nM and 198 nM, respectively. SIGNIFICANCE: The calix[4]arene macrocycle's excellent size and binding cone conformation make it a good host-guest interface for the pyrophosphate anion and ADP. The bis-ruthenium polypyridyl complex's connection to the p-tertbutyl calix[4]arene moiety creates the ADP selectivity turn-on sensor. When moving from mono-nuclear to bi-nuclear ruthenium complex anchored on p-tertbutyl calix[4]arene, the probe can differentiate ADP, ATP, and AMP. Furthermore, this platform is a great resource for creating devices to simultaneously assess phosphate anions in environmental samples.

Dual-signal fluorescence aptasensing system for adenosine triphosphate assisting by MoS2 nanosheets.

Adenosine triphosphate (ATP) has an irreplaceable role in the maintenance of many physiological processes and biological functions, and can be employed as an indicator of many diseases. In this work, we constructed a simple and sensitive dual-signal fluorescence aptasensing system for ATP detection with berberine as the signal reporter, ATP-aptamer as the recognition unit and MoS2 nanosheets as the signal amplification. In the absence of ATP, berberine can bind to the single-stranded DNA (ssDNA) of ATP-aptamer and selectively assemble on the surface of MoS2 nanosheets, leading to the fluorescence quenching of bererbine based on the fluorescence resonance energy transfer, denoted by "OFF". Accordingly, the fluorescence anisotropy signal is enhanced due to restriction on rotate of the fluorescent probe and denoted as "ON". Conversely, in the presence of ATP, it specifically interacts with ATP-aptamer and switches the free-curled single-stranded of ATP-aptamer to the G-quadruplex structure of ATP-aptamer/ATP/berberine, causing the detachment from the surface of the MoS2 nanosheet. Accordingly, the fluorescence signal was reversed from "OFF" to "ON", and the fluorescence anisotropy signal was turned "ON" to "OFF". The developed aptasensing system achieved a desirable sensitivity of 40.0 nM with fluorescent mode, and of 20.8 nM with fluorescent anisotropic mode. The sensing system has demonstrated high quality detection performance in human serum sample, and obtained the satisfactory recovery results for fluorescent of 93.0-108.5%, fluorescent anisotropic of 96.4-106.7%.

Bioenergetic effects of pristine and ultraviolet-weathered polydisperse polyethylene terephthalate and polystyrene nanoplastics on human intestinal Caco-2 cells.

The ubiquitous human exposure to nanoplastics (NPs) increasingly raises concerns regarding impact on our health. However, little is known on the biological effects of complex mixtures of weathered NPs with heterogenous size and irregular shape present in the environment. In this study, the bioenergetic effects of four such NPs mixtures on human intestinal Caco-2 cells were investigated. To this aim, Caco-2 cells were exposed to polydisperse nanoPET (<800 nm) and nanoPS (mixture of 100 and 750 nm) samples with and without ultraviolet (UV) weathering at low concentration range (102-107 particles/mL) for 48 h. Mitochondrial respiration, glycolytic functions and ATP production rates of exposed cells were measured by Seahorse XFe96 Analyzer. Among four NPs samples, polydisperse nanoPET with irregular shapes induced significant stimulation of mitochondrial respiration, glycolysis and ATP production rates in Caco-2 cells. Spherical nanoPS caused significant stimulation on glycolytic functions of Caco-2 cells at the highest concentration used (106 particles/mL). ATR-FTIR spectra and carbonyl index indicated formation of carbonyl groups in nanoPET and nanoPS after UV weathering. UV weathering could alleviate bioenergetic stress caused by NPs in Caco-2 cells and even shifted the energy pathways from mitochondrial respiration to glycolysis due to electrostatic repulsion between negatively charged UV-aged NPs and cell membranes. This research is the first to study in-vitro bioenergetic responses of NPs samples with multidimensional features (polymer type, irregular shape, heterogenous size, UV-weathering) on human health. It highlights that effects between pristine and weathered NPs are different at a bioenergetic level, which has important implications for the risk assessment of NPs on human health.

Crucial physicochemical factors mediating mitochondrial toxicity of nanoparticles at noncytotoxic concentration.

Nanomaterials have been extensively applied in multiple industries, among which silver nanoparticles (AgNPs), silicon dioxide nanoparticles (SiNPs), and gold nanoparticles (AuNPs) have become representative of widely consumed NPs. Limited knowledge is available regarding the subcellular responses of NPs with different physicochemical properties, i.e. material type and size, under the noncytotoxic concentrations. Macrophages are important sensitive cells exposed to NPs, and mitochondria are sensitive organelles that respond at the subcellular level. Herein, we found that sublethal concentrations of AgNPs and SiNPs, not AuNPs, decreased the mitochondrial membrane potential (MMP) and tubular mitochondria, and further resulted in an increase of ROS level and a decrease of ATP generation. AgNPs and SiNPs can also disturb mitochondrial dynamics manifested as increasing Mfn2 expression and decreasing Drp1 expression. Further assessments for mitochondrial function showed that AgNPs and SiNPs exposure led to a decrease in the gene expressions related to complex I (Ndufa8 and Ndufs2), complex III (Uqcrc2 and Uqcrfs1), complex IV (Cox6b1), and activity of complex I, suggesting their potential roles in impairing cellular respiration. In terms of the effects of NPs with different sizes, stronger toxicity was observed in smaller-sized nanoparticles. Among the above mitochondrial changes, we identified that ROS, ATP, MMP, tubular mitochondria, and expression of Drp1 were relatively sensitive indicators in subcellular response to NPs. With the above sensitive indicators, the comparison of heterogeneity between material type and size of the NPs showed that material type occupied a main influence on subcellular mitochondrial effects. Our finding provided important data on the potential subcellular risks of NPs, and indicated the vital role of material type for a better understanding of the nanomaterial biological safety.

Mechanism of DaiTongXiao in the treatment of gouty arthritis through the NLRP3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: DaiTongXiao (DTX) is a traditional Chinese Dai folk formulation utilized for gouty arthritis treatment, with substantial evidence supporting its anti-inflammatory properties. The NLRP3 inflammasome disorder is tightly linked to the development of many inflammatory diseases. AIM OF THE STUDY: To elucidate the therapeutic efficacy of DTX in gouty arthritis and reveal its potential underlying mechanism. MATERIALS AND METHODS: The primary active constituents in DTX were determined through ultraviolet spectrophotometry and gas chromatography. Rats underwent induction with monosodium urate (MSU), followed by treatment of J774A.1 cells with adenosine triphosphate (ATP) activation and lipopolysaccharide (LPS) induction and the subsequent culture in Dulbecco's modified Eagle's medium. The degree of foot joint swelling in rats was assessed, and ankle joints were evaluated through H&E staining. Enzyme-linked immunosorbent assay was performed to measure the levels of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α in both serum and cells. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the relative mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 macrophages. The expression of NLRP3, ASC, Caspase-1, and NF-κB was examined by western blotting. RESULTS: DTX could alleviate MSU-induced joint swelling in rats, as evidenced by a reduction in joint inflammation. Moreover, DTX effectively enhanced the survival rate of J774A.1 cells following LPS induction and ATP activation. Furthermore, DTX significantly reduced IL-1ß, IL-6, IL-8, and TNF-α levels in both cell culture medium and rat serum. RT-PCR results revealed that DTX notably downregulated the mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 cells. Additionally, DTX downregulated NLRP3, ASC, NF-κB, and Caspase-1 expression in the joint tissue. CONCLUSIONS: DTX exerts a significant anti-gouty arthritis effect, with its mechanism being tightly linked to the NLRP3 inflammatory signaling pathway. This pathway may be modulated by inhibiting IL-1ß differentiation and maturation by downregulating NLRP3, ASC, Caspase-1, and NF-κB protein expression. This, in turn, leads to a reduction in the release of IL-6, IL-8, and TNF-α, ultimately impeding gouty arthritis progression.

The multifaceted role of extracellular ATP in sperm function: From spermatogenesis to fertilization.

Extracellular adenosine 5'-triphosphate (ATP) is a vital signaling molecule involved in various physiological processes within the body. In recent years, studies have revealed its significant role in male reproduction, particularly in sperm function. This review explores the multifaceted role of extracellular ATP in sperm function, from spermatogenesis to fertilization. We discuss the impact of extracellular ATP on spermatogenesis, sperm maturation and sperm-egg fusion, highlighting the complex regulatory mechanisms and potential clinical applications in the context of male infertility. By examining the latest research, we emphasize the crucial role of extracellular ATP in sperm function and propose future research directions to further.

Calcium electroporation causes ATP depletion in cells and is effective both in microsecond and nanosecond pulse range as a modality of electrochemotherapy.

Calcium electroporation is a modality of electrochemotherapy (ECT), which is based on intracellular electric field-mediated delivery of cytotoxic doses of calcium into the cells resulting in rapid cell death. In this work, we have developed a CHO-K1 luminescent cell line, which allowed the estimation of cell membrane permeabilization, ATP depletion and cytotoxicity evaluation without the use of additional markers and methodologies. We have shown the high efficiency of nanosecond pulses compressed into a MHz burst for application in calcium ECT treatments. The 5 kV/cm and 10 kV/cm nanosecond (100 and 600 ns) pulses were delivered in bursts of 10, 50 and 100 pulses (a total of 12 parametric protocols) and then compared to standard microsecond range sequences (100 µs × 8) of 0.4-1.4 kV/cm. The effects of calcium-free, 2 mM and 5 mM calcium electroporation treatments were characterized. It was shown that reversible electroporation is accompanied by ATP depletion associated with membrane damage, while during calcium ECT the ATP depletion is several-fold higher, which results in cell death. Finally, efficacy-wise equivalent pulse parameters from nanosecond and microsecond ranges were established, which can be used for calcium nano-ECT as a better alternative to ESOPE (European Standard Operating Procedures on Electrochemotherapy) protocols.

A voltammetric study of nitrogenase MoFe-protein using low-potential electron transfer mediators.

The molybdenum-iron protein (MoFeP), a component of the enzyme nitrogenase, catalyzes the reduction of an array of small molecules, including N2 to NH3. In microorganisms, during the catalytic cycle, MoFeP receives electrons from the obligate biological redox partner iron protein (FeP) in a process coupled to the hydrolysis of two MgATP per one electron transferred. Despite the favorable redox properties of the cofactors, the requirement of the MgATP hydrolysis significantly decreases the energy efficiency of MoFeP. Therefore, remarkable efforts have been devoted to electrochemically activating MoFeP without FeP and MgATP. Previously, MoFeP was adsorbed on an electrode surface and revealed a slow catalysis with and without electron transfer mediators. However, enzyme adsorption can cause conformational and structural changes in a fragile protein molecule and alter its catalytic activity. In this work, MoFeP was electrochemically studied in solution. Various electron transfer mediators with potentials ranging from -0.3 V to -1 V (vs. NHE) were examined with MoFeP using cyclic voltammetry. No significant catalytic activity of the MoFeP was observed with any of the tested mediators. This indicates that efficient electrochemical activation of MoFeP cannot be achieved exclusively by increasing the driving force between the MoFeP redox cofactors and an electron donor.

Electrolysis products, reactive oxygen species and ATP loss contribute to cell death following irreversible electroporation with microsecond-long pulsed electric fields.

Membrane permeabilization and thermal injury are the major cause of cell death during irreversible electroporation (IRE) performed using high electric field strength (EFS) and small number of pulses. In this study, we explored cell death under conditions of reduced EFS and prolonged pulse application, identifying the contributions of electrolysis, reactive oxygen species (ROS) and ATP loss. We performed ablations with conventional high-voltage low pulse (HV-LP) and low-voltage high pulse (LV-HP) conditions in a 3D tumor mimic, finding equivalent ablation volumes when using 2000 V/cm 90 pulses or 1000 V/cm 900 pulses respectively. These results were confirmed by performing ablations in swine liver. In LV-HP treatment, ablation volume was found to increase proportionally with pulse numbers, without the substantial temperature increase seen with HV-LP parameters. Peri-electrode pH changes, ATP loss and ROS production were seen in both conditions, but LV-HP treatments were more sensitive to blocking of these forms of cell injury. Increases in current drawn during HV-LP was not observed during LV-HP condition where the total ablation volume correlated to the charge delivered into the tissue which was greater than HV-LP treatment. LV-HP treatment provides a new paradigm in using pulsed electric fields for tissue ablation with clinically relevant volumes.

Real-Time Assessment of Mitochondrial Function in Cytotrophoblast and Syncytialized Trophoblast Cells Using the Seahorse XFe24 Extracellular Flux Analyzer.

Physiological processes utilize variable amounts of energy required for optimal growth, development, and survival. This energy is supplied by total intracellular adenosine triphosphate (ATP) and is mainly generated by mitochondrial oxidative phosphorylation and to a lesser extent via glycolysis. Here, we provide a detailed protocol for obtaining measurements of energy metabolism using the Seahorse XFe24 Extracellular Flux Analyzer. Specifically, this assay measures mitochondrial oxidative phosphorylation based on oxygen consumption rate (OCR) and glycolysis by analyzing the extracellular acidification rate (ECAR) via real-time live cell analysis. Using trophoblast cell lines, this protocol focuses on analyzing mitochondrial respiration for both cytotrophoblasts and syncytiotrophoblasts.

Evaluation of light irradiation on anthocyanins and energy metabolism of grape (Vitis vinifera L.) during storage.

Grape is the world's economic horticultural crop; it is perishable due to various pathogens and abiotic stress attributed to water loss-induced issues. To address these postharvest problems, this research investigates the effects of light irradiation on anthocyanins synthesis and energy metabolism in stored grapes to enhance their postharvest quality. The activities of chlorophyllase (1.17 U gk-1), Mg-dechelatase (351.69 U gk-1), chlorophyll-degraded peroxidase (3.49 U gk-1), and pheophytinase (0.85 U gk-1) were significantly higher in the control fruit than in the treated fruit at the end of storage. The red-light treatment showed higher levels of anthocyanins biosynthesis-related enzymes than green, blue-light, and control treatments. Additionally, light irradiation resulted in a decrease in adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and energy charge. This was attributed to decreased activity of energy metabolism enzymes, reduced nicotinamide adenine dinucleotide phosphate content, and increased nicotinamide adenine dinucleotide content. These findings offer a theoretical foundation for optimizing grape coloration and energy metabolism during storage, thus prolonging the shelf-life of grapes by improving quality attributes. This research highlights the potential of light irradiation as a technique for enhancing the postharvest quality of agricultural products.

Neurotoxicity study of ethyl acetate extract of Zanthoxylum armatum DC. on SH-SY5Y based on ROS mediated mitochondrial apoptosis pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC. (ZADC) is a traditional medicinal plant with various pharmacological activities and is widely used in China, Japan, India, and other regions. Previous studies have revealed that the methanol extract of ZADC can cause neurotoxicity symptoms in rats, such as drooling, decreased appetite, decreased movement, and increased respiratory rate. However, the basis of these toxic substances and the mechanism of neurotoxicity remain unclear. AIM OF THE STUDY: To evaluate the effects of ZADC on nerve cells and their damage mechanisms and discuss the possible toxic substance basis. MATERIALS AND METHODS: The ethyl acetate extract of ZADC is obtained by extracting the methanol extract of ZADC with ethyl acetate. The Q-Orbitrap LC-MS/MS method was employed to analyze the chemical composition of the EA extract of ZADC. SH-SY5Y cells were incubated with different concentrations of the ethyl acetate extract of ZADC. The cytotoxicity of the extract was evaluated using CCK-8, LDH, and ROS assays, and the oxidative stress status of cells was assessed using MDA, GSH, and SOD. Cell apoptosis was detected using flow cytometry. Damage to mitochondrial function was evaluated by labeling mitochondria, ATP, and MMP with fluorescence. Cyto-C, Caspase-3, Caspase-9, Apaf-1, Bax, and reduced Bcl2 expression were measured to evaluate the activation of the mitochondrial apoptosis pathway. Finally, NAC intervention was used to detect changes in the relevant indicators. The activation of mitochondrial apoptosis pathway was evaluated by measuring Cyto-C, Caspase-3, Caspase-9, Apaf-1, and Bax and Bcl2 expression. Finally, NAC intervention was utilized to detect changes in the relevant indicators. RESULTS: After treating SY-SY5Y cells with EA extract from ZADC, cell viability decreased significantly, and the intracellular ROS level increased in a dose-dependent manner. Meanwhile, ZADC can cause cellular oxidative stress and increase MDA and SOD concentrations while decreasing GSH concentrations. It can also shorten the mitochondrial cristae and decrease the number of mitochondria. In contrast, it can reduce ATP synthesis in the mitochondria and mitochondrial membrane potential (MMP). Furthermore, it increased the apoptosis rate and the expression of Cyto-C, Caspase-3, Caspase-9, Apaf-1, and Bax and reduced Bcl2 expression. NAC intervention alleviated the reduction in SH-SY5Y cell survival and the accumulation of reactive oxygen species induced by the EA extract in ZADC. It also inhibits signaling pathways dominated by proteins, such as Cyto-C, reducing cell apoptosis and cytotoxicity. A total of 46 compounds were identified in the extracts. CONCLUSIONS: The results suggest that EA extract of ZADC can induce the mitochondrial apoptotic pathway by accumulating ROS in cells, leading to apoptosis. Antioxidants had a good inhibitory and protective effect against cell damage caused by the EA extract of ZADC. The neurotoxic components of ZADC may be organic acids and compounds containing amino groups.