Nanoelectrochemistry reveals how presynaptic neurons regulate vesicle release to sustain synaptic plasticity under repetitive stimuli.

Synaptic plasticity is the ability of synapses to modulate synaptic strength in response to dynamic changes within, as well as environmental changes. Although there is a considerable body of knowledge on protein expression and receptor migration in different categories of synaptic plasticity, the contribution and impact of presynaptic vesicle release and neurotransmitter levels towards plasticity remain largely unclear. Herein, nanoelectrochemistry using carbon fiber nanoelectrodes with excellent spatio-temporal resolution was applied for real-time monitoring of presynaptic vesicle release of dopamine inside single synapses of dopaminergic neurons, and exocytotic variations in quantity and kinetics under repetitive electrical stimuli. We found that the presynaptic terminal tends to maintain synaptic strength by rapidly recruiting vesicles, changing the dynamics of exocytosis, and maintaining sufficient neurotransmitter release in following stimuli. Except for small clear synaptic vesicles, dense core vesicles are involved in exocytosis to sustain the neurotransmitter level in later periods of repetitive stimuli. These data indicate that vesicles use a potential regulatory mechanism to establish short-term plasticity, and provide new directions for exploring the synaptic mechanisms in connection and plasticity.

Nanoelectrochemistry monitoring of intracellular reactive oxygen and nitrogen species induced by nanoplastic exposure.

Airborne nanoplastics can enter alveolar cells and trigger intracellular oxidative stress primarily. Herein, taking advantage of the high electrochemical resolution of SiC@Pt nanoelectrodes, we achieved the quantitative discrimination of the major ROS/RNS within A549 cells, disclosed the sources of their precursors, and observed that the NO (RNS precursor) level significantly increased, whereas O2˙- (ROS precursor) remained relatively stable during the nanoplastics exposure. This establishes that iNOS or mitochondrion-targeted treatment may be a preventive or therapeutic strategy for nanoplastic-induced lung injury.

Real-Time Quantification of Nanoplastics-Induced Oxidative Stress in Stretching Alveolar Cells.

Nanoplastics from air pollutants can be directly inhaled into the alveoli in the lungs and further enter blood circulation, and numerous studies have revealed the close relation between internalized nanoplastics with many physiological disorders via intracellular oxidative stress. However, the dynamic process of nanoplastics-induced oxidative stress in lung cells under breath-mimicked conditions is still unclear, due to the lack of methods that can reproduce the mechanical stretching of the alveolar and simultaneously monitor the oxidative stress response. Here, we describe a biomimetic platform by culturing alveoli epithelial cells on a stretchable electrochemical sensor and integrating them into a microfluidic device. This allows reproducing the respiration of alveoli by cyclic stretching of the alveoli epithelial cells and monitoring the nanoplastics-induced oxidative stress by the built-in sensor. By this device, we prove that cyclic stretches can greatly enhance the cellular uptake of nanoplastics with the dependencies of strain amplitude. Importantly, oxidative stress evoked by internalized nanoplastics can be quantitatively monitored in real time. This work will promote the deep understanding about the cytotoxicity of inhaled nanoplastics in the pulmonary mechanical microenvironment.

Nanosensor detection of reactive oxygen and nitrogen species leakage in frustrated phagocytosis of nanofibres.

Exposure to widely used inert fibrous nanomaterials (for example, glass fibres or carbon nanotubes) may result in asbestos-like lung pathologies, becoming an important environmental and health concern. However, the origin of the pathogenesis of such fibres has not yet been clearly established. Here we report an electrochemical nanosensor that is used to monitor and quantitatively characterize the flux and dynamics of reactive species release during the frustrated phagocytosis of glass nanofibres by single macrophages. We show the existence of an intense prolonged release of reactive oxygen and nitrogen species by single macrophages near their phagocytic cups. This continued massive leakage of reactive oxygen and nitrogen species damages peripheral cells and eventually translates into chronic inflammation and lung injury, as seen during in vitro co-culture and in vivo experiments.

Multi-scenario optimization of land use structure and prediction of ecosystem service value in Guanzhong Plain urban agglomeration.

Rapid economic development has led to significant changes in land use in the Guanzhong Plain urban agglomeration, which alters regional ecosystem service value (ESV). Based on the land use and driver data of the Guanzhong Plain urban agglomeration, we used the system dynamics (SD) model coupled with the mixed-cell cellular automata (MCCA) model to predict the subtle spatial and temporal changes of ESV within the land use unit in 2040 under the scenarios of natural development, economic development, ecological protection, and arable land conservation, to reveal the responses of ESV to the socio-economic evolution. We examined the impacts of land use change on ESV by using the sensitivity index. The results showed that land use transformation between 2000 and 2020 in the study area was mainly the conversion between arable land, forest, grassland, and the conversion of arable land to construction land. Due to increased forest and water, ESV increased slightly during this period. In 2040, compared with the ecological protection scenario, the proportion of forest in the economic development scenario decreased by 1.8%, and the construction land increased by 1.3%. During 2020-2040, under the economic development scenario, ESV showed a downward trend in the central and eastern regions but an upward trend under the arable land conservation scenario, with hydrological and climatic regulation contributing the most to ESV. Total ESV showed a decreasing trend except for the ecological conservation scenario. In the ecological protection scenario, land use change positively impacted ESV. In contrast, ESV had a negative response to land use change in other scenarios, with the greatest reduction in the economic development scenario. The research could provide new methods for multi-scenario land use simulation and ESV prediction and have scientific and practical significance for optimizing land space layout, land resource planning management, and sustainable development path strategy of urban agglomerations.

Enhanced BCAT1 activity and BCAA metabolism promotes RhoC activity in cancer progression.

Increased expression of branched-chain amino acid transaminase 1 or 2 (BCAT1 and BCAT2) has been associated with aggressive phenotypes of different cancers. Here we identify a gain of function of BCAT1 glutamic acid to alanine mutation at codon 61 (BCAT1E61A) enriched around 2.8% in clinical gastric cancer samples. We found that BCAT1E61A confers higher enzymatic activity to boost branched-chain amino acid (BCAA) catabolism, accelerate cell growth and motility and contribute to tumor development. BCAT1 directly interacts with RhoC, leading to elevation of RhoC activity. Notably, the BCAA-derived metabolite, branched-chain α-keto acid directly binds to the small GTPase protein RhoC and promotes its activity. BCAT1 knockout-suppressed cell motility could be rescued by expressing BCAT1E61A or adding branched-chain α-keto acid. We also identified that candesartan acts as an inhibitor of BCAT1E61A, thus repressing RhoC activity and cancer cell motility in vitro and preventing peritoneal metastasis in vivo. Our study reveals a link between BCAA metabolism and cell motility and proliferation through regulating RhoC activation, with potential therapeutic implications for cancers.

Dual-channel nanoelectrochemical sensor for monitoring intracellular ROS and NADH kinetic variations of their concentrations.

Reactive oxygen species (ROS) and nicotinamide adenine dinucleotide (NADH) are important intracellular redox-active molecules involved in various pathological processes including inflammation, neurodegenerative diseases, and cancer. However, the fast dynamic changes and mutual regulatory kinetic relationship between intracellular ROS and NADH in these biological processes are still hard to simultaneously investigate. A dual-channel nanowire electrode (DC-NWE) integrating two conductive nanowires, one functionalized with platinum nanoparticles and the other with conductive polymer, was nanofabricated for the selective and simultaneous real-time monitoring of intracellular ROS and NADH release by mitochondria in single living MCF-7 tumoral cells stimulated by resveratrol. The production of ROS was observed to occur tenths of a second before the release of NADH, a significant new piece of information suggesting a mechanism of action of resveratrol. Beyond the importance of the specific data gathered in this study, this work established the feasibility of simultaneously monitoring multiple species and analyzing their kinetics relationships over sub-second time scales thanks to dual-channel nanowire electrodes. It is believed that this concept and its associated nanoelectrochemical tools might benefit to a deeper understanding of mutual regulatory relationship between intracellular crucial molecular markers during physiological and pathological processes as well as for evaluating medical treatments.

Homeostasis inside Single Activated Phagolysosomes: Quantitative and Selective Measurements of Submillisecond Dynamics of Reactive Oxygen and Nitrogen Species Production with a Nanoelectrochemical Sensor.

Reactive oxygen and nitrogen species (ROS/RNS) are generated by macrophages inside their phagolysosomes. This production is essential for phagocytosis of damaged cells and pathogens, i.e., protecting the organism and maintaining immune homeostasis. The ability to quantitatively and individually monitor the four primary ROS/RNS (ONOO-, H2O2, NO, and NO2-) with submillisecond resolution is clearly warranted to elucidate the still unclear mechanisms of their rapid generation and to track their concentration variations over time inside phagolysosomes, in particular, to document the origin of ROS/RNS homeostasis during phagocytosis. A novel nanowire electrode has been specifically developed for this purpose. It consisted of wrapping a SiC nanowire with a mat of 3 nm platinum nanoparticles whose high electrocatalytic performances allow the characterization and individual measurements of each of the four primary ROS/RNS. This allowed, for the first time, a quantitative, selective, and statistically robust determination of the individual amounts of ROS/RNS present in single dormant phagolysosomes. Additionally, the submillisecond resolution of the nanosensor allowed confirmation and measurement of the rapid ability of phagolysosomes to differentially mobilize their enzyme pools of NADPH oxidases and inducible nitric oxide synthases to finely regulate their homeostasis. This reveals an essential key to immune responses and immunotherapies and rationalizes its biomolecular origin.

Large-Scale Synthesis of Functionalized Nanowires to Construct Nanoelectrodes for Intracellular Sensing.

A strategy for one-pot and large-scale synthesis of functionalized core-shell nanowires (NWs) to high-efficiently construct single nanowire electrodes is proposed. Based on the polymerization reaction between 3,4-ethylenedioxythiophene (EDOT) and noble metal cations, manifold noble metal nanoparticles-polyEDOT (PEDOT) nanocomposites can be uniformly modified on the surface of any nonconductive NWs. This provides a facile and versatile approach to produce massive number of core-shell NWs with excellent conductivity, adjustable size, and well-designed properties. Nanoelectrodes manufactured with such core-shell NWs exhibit excellent electrochemical performance and mechanical stability as well as favorable antifouling properties, which are demonstrated by in situ intracellular monitoring of biological molecules (nitric oxide) and unraveling its relevant unclear signaling pathway inside single living cells.

Intracellular monitoring of NADH release from mitochondria using a single functionalized nanowire electrode.

Mitochondria are the powerhouse of cells, and also their suicidal weapon store. Mitochondrial dysfunction can cause the opening of the mitochondrial permeability transition pore (mPTP) and nicotinamide adenine dinucleotide (NADH) release from mitochondria, eventually leading to the disruption of energy metabolism and even cell death. Hence, NADH is often considered a marker of mitochondrial function, but in situ monitoring of NADH release from mitochondria in single living cells remains a great challenge. Herein, we develop a functionalized single nanowire electrode (NWE) for electrochemical detection of NADH release from intracellular mitochondria by modifying conductive polymer (poly(3,4-ethylendioxythiophene), PEDOT)-coated carbon nanotubes (CNTs) on the surface of a SiC@C nanowire. The positively charged PEDOT facilitates the accumulation of negatively charged NADH at the electrode surface and CNTs promote electron transfer, thus endowing the NWE with high sensitivity and selectivity. Further studies show that resveratrol, a natural product, specifically induced NADH release from mitochondria of MCF-7 cancer cells rather than non-cancerous MCF-10 A cells, indicating the potential therapeutic effects of resveratrol in cancer treatment. This work provides an efficient method to monitor mitochondrial function by in situ electrochemical measurement of NADH release, which will be of great benefit for physiological and pathological studies.