Vitamin C Drives Reentrant Actin Phase Transition: Biphasic Exocytosis Regulation Revealed by Single-Vesicle Electrochemistry.

Unveiling molecular mechanisms that dominate protein phase dynamics has been a pressing need for deciphering the intricate intracellular modulation machinery. While ions and biomacromolecules have been widely recognized for modulating protein phase separations, effects of small molecules that essentially constitute the cytosolic chemical atmosphere on the protein phase behaviors are rarely understood. Herein, we report that vitamin C (VC), a key small molecule for maintaining a reductive intracellular atmosphere, drives reentrant phase transitions of myosin II/F-actin (actomyosin) cytoskeletons. The actomyosin bundle condensates dissemble in the low-VC regime and assemble in the high-VC regime in vitro or inside neuronal cells, through a concurrent myosin II protein aggregation-dissociation process with monotonic VC concentration increase. Based on this finding, we employ in situ single-cell and single-vesicle electrochemistry to demonstrate the quantitative modulation of catecholamine transmitter vesicle exocytosis by intracellular VC atmosphere, i.e., exocytotic release amount increases in the low-VC regime and decreases in the high-VC regime. Furthermore, we show how VC regulates cytomembrane-vesicle fusion pore dynamics through counteractive or synergistic effects of actomyosin phase transitions and the intracellular free calcium level on membrane tensions. Our work uncovers the small molecule-based reversive protein phase regulatory mechanism, paving a new way to chemical neuromodulation and therapeutic repertoire expansion.

Confining Thiolysis of Dinitrophenyl Ether to a Luminescent Metal-Organic Framework with a Large Stokes Shift for Highly Efficient Detection of Hydrogen Sulfide in Rat Brain.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates various physiological and pathological processes in the central nervous system. It is vital to develop an effective method to detect H2S in vivo to elucidate its critical role. However, current fluorescent probes for accurate quantification of H2S still face big challenges due to complicated fabrication, small Stokes shift, unsatisfactory selectivity, and especially delayed response time. Herein, based on simple postsynthetic modification, we present an innovative strategy by confining H2S-triggered thiolysis of dinitrophenyl (DNP) ether within a luminescent metal-organic framework (MOF) to address those issues. Due to the cleavage of the DNP moiety by H2S, the nanoprobe gives rise to a remarkable fluorescence turn-on signal with a large Stokes shift of 190 nm and also provides high selectivity to H2S against various interferents including competing biothiols. In particular, by virtue of the unique structural property of the MOF, it exhibits an ultrafast sensing ability for H2S (only 5 s). Moreover, the fluorescence enhancement efficiency displays a good linear correlation with H2S concentration in the range of 0-160 µM with a detection limit of 0.29 µM. Importantly, these superior sensing performances enable the nanoprobe to measure the basal value and monitor the change of H2S level in the rat brain.

Enzymatic Galvanic Redox Potentiometry for In Vivo Biosensing.

Redox potentiometry has emerged as a new platform for in vivo sensing, with improved neuronal compatibility and strong tolerance against sensitivity variation caused by protein fouling. Although enzymes show great possibilities in the fabrication of selective redox potentiometry, the fabrication of an enzyme electrode to output open-circuit voltage (EOC) with fast response remains challenging. Herein, we report a concept of novel enzymatic galvanic redox potentiometry (GRP) with improved time response coupling the merits of the high selectivity of enzyme electrodes with the excellent biocompatibility and reliability of GRP sensors. With a glucose biosensor as an illustration, we use flavin adenine dinucleotide-dependent glucose dehydrogenase as the recognition element and carbon black as the potential relay station to improve the response time. We find that the enzymatic GRP biosensor rapidly responds to glucose with a good linear relationship between EOC and the logarithm of glucose concentration within a range from 100 µM to 2.65 mM. The GRP biosensor shows high selectivity over O2 and coexisting neurochemicals, good reversibility, and sensitivity and can in vivo monitor glucose dynamics in rat brain. We believe that this study will pave a new platform for the in vivo potentiometric biosensing of chemical events with high reliability.

Ion-Selective Micropipette Sensor for In Vivo Monitoring of Sodium Ion with Crown Ether-Encapsulated Metal-Organic Framework Subnanopores.

In vivo sensing of the dynamics of ions with high selectivity is essential for gaining molecular insights into numerous physiological and pathological processes. In this work, we report an ion-selective micropipette sensor (ISMS) through the integration of functional crown ether-encapsulated metal-organic frameworks (MOFs) synthesized in situ within the micropipette tip. The ISMS features distinctive sodium ion (Na+) conduction and high selectivity toward Na+ sensing. The selectivity is attributed to the synergistic effects of subnanoconfined space and the specific coordination of 18-crown-6 toward potassium ions (K+), which largely increase the steric hindrance and transport resistance for K+ to pass through the ISMS. Furthermore, the ISMS exhibits high stability and sensitivity, facilitating real-time monitoring of Na+ dynamics in the living rat brain during spreading of the depression events process. In light of the diversity of crown ethers and MOFs, we believe this study paves the way for a nanofluidic platform for in vivo sensing and neuromorphic electrochemical sensing.

Nonthermal and reversible control of neuronal signaling and behavior by midinfrared stimulation.

Various neuromodulation approaches have been employed to alter neuronal spiking activity and thus regulate brain functions and alleviate neurological disorders. Infrared neural stimulation (INS) could be a potential approach for neuromodulation because it requires no tissue contact and possesses a high spatial resolution. However, the risk of overheating and an unclear mechanism hamper its application. Here we show that midinfrared stimulation (MIRS) with a specific wavelength exerts nonthermal, long-distance, and reversible modulatory effects on ion channel activity, neuronal signaling, and sensorimotor behavior. Patch-clamp recording from mouse neocortical pyramidal cells revealed that MIRS readily provides gain control over spiking activities, inhibiting spiking responses to weak inputs but enhancing those to strong inputs. MIRS also shortens action potential (AP) waveforms by accelerating its repolarization, through an increase in voltage-gated K+ (but not Na+) currents. Molecular dynamics simulations further revealed that MIRS-induced resonance vibration of -C=O bonds at the K+ channel ion selectivity filter contributes to the K+ current increase. Importantly, these effects are readily reversible and independent of temperature increase. At the behavioral level in larval zebrafish, MIRS modulates startle responses by sharply increasing the slope of the sensorimotor input-output curve. Therefore, MIRS represents a promising neuromodulation approach suitable for clinical application.

Life history traits of low-toxicity alternative bisphenol S on Daphnia magna with short breeding cycles: A multigenerational study.

Due to relatively lower toxicity, bisphenol S (BPS) has become an alternative to previously used bisphenol A. Nevertheless, the occurrence of BPS and its ecological impact have recently attracted increasing attentions because the toxicology effect of BPS with life cycle or multigenerational exposure on aquatic organisms remains questionable. Herein, Daphnia magna (D. magna) multigenerational bioassays spanning four generations (F0-F3) and single-generation recovery (F1 and F3) in clean water were used to investigate the ecotoxicology of variable chronic BPS exposure. For both assays, four kinds of life-history traits (i.e., survival, reproduction, growth and ecological behavior) were examined for each generation. After an 18-day exposure under concentration of 200 µg/L, the survival rate of D. magna was less than 15 % for the F2 generation, whereas all died for the F3 generation. With continuous exposure of four generations of D. magna at environmentally relevant concentrations of BPS (2 µg/L), inhibition of growth and development, prolonged sexual maturity, decreased offspring production and decreased swimming activity were observed for the F3 generation. In particular, it is difficult for D. magna to return to its normal level through a single-generation recovery in clean water in terms of reproductive function, ecological behavior and population health. Hence, multi-generational exposure to low concentrations of BPS can have adverse effects on population health of aquatic organisms with short breeding cycles, highlighting the necessity to assess the ecotoxicology of chronic BPS exposure for public health.

The Pathogenesis in Alzheimer's Disease: TREM2 as a Potential Target.

Alzheimer's disease (AD) is ranked as the third-most expensive illness and sixth leading cause of mortality. It is associated with the deposition of extracellular amyloid-ß (Aß) in neural plaques (NPs), as well as intracellular hyperphosphorylated tau proteins that form neurofibrillary tangles (NFTs). As a new target in regulating neuroinflammation in AD, triggering receptor expressed on myeloid cells 2 (TREM2) is highly and exclusively expressed on the microglial surface. TREM2 interacts with adaptor protein DAP12 to initiate signal pathways that mainly dominant microglia phenotype and phagocytosis mobility. Furthermore, TREM2 gene mutations confer increased AD risk, and TREM2 deficiency exhibits more dendritic spine loss around neural plaques. Mechanisms for regulating TREM2 to alleviate AD has evolved as an area of AD research in recent years. Current medications targeting Aß or tau proteins are unable to reverse AD progression. Emerging evidence implicating neuroinflammation may provide novel insights, as early microglia-related inflammation can be induced decades prior to the commencement of AD-related cognitive damage. Physical exercise can exert a neuroprotective effect over the course of AD progression. This review aims to (1) summarize the pathogenesis of AD and recent updates in the field, (2) assess the concept that AD cognitive impairment is closely correlated with microglia-related inflammation, and (3) review TREM2 functions and its role between exercise and AD, which is likely to be an ideal candidate target.

Galvanic Redox Potentiometry for Fouling-Free and Stable Serotonin Sensing in a Living Animal Brain.

Serotonin (5-HT) is a major neurotransmitter broadly involved in many aspects of feeling and behavior. Although its electro-activity makes it a promising candidate for electrochemical sensing, the persistent generation of fouling layers on the electrode by its oxidation products presents a hurdle for reliable sensing. Here, we present a fouling-free 5-HT sensor based on galvanic redox potentiometry. The sensor efficiently minimizes electrode fouling as revealed by in situ Raman spectroscopy, ensuring a less than 3 % signal change in a 2 hour continuous experiment, whereas amperometric sensors losing 90 % within 30 min. Most importantly, the sensor is highly amenable for in vivo studies, permitting real-time 5-HT monitoring, and supporting the mechanism associated with serotonin release in brain. Our system offers an effective way for sensing different neurochemicals having significant fouling issues, thus facilitating the molecular-level understanding of brain function.

Direct Quantification of Nanoplastics Neurotoxicity by Single-Vesicle Electrochemistry.

Nanoplastics are recently recognized as neurotoxic factors for the nervous systems. However, whether and how they affect vesicle chemistry (i.e., vesicular catecholamine content and exocytosis) remains unclear. This study offers the first direct evidence for the nanoplastics-induced neurotoxicity by single-vesicle electrochemistry. We observe the cellular uptake of polystyrene (PS) nanoplastics into model neuronal cells and mouse primary neurons, leading to cell viability loss depending on nanoplastics exposure time and concentration. By using single-vesicle electrochemistry, we find the reductions in the vesicular catecholamine content, the frequency of stimulated exocytotic spikes, the neurotransmitter release amount of single exocytotic event, and the membrane-vesicle fusion pore opening-closing speed. Mechanistic investigations suggest that PS nanoplastics can cause disruption of filamentous actin (F-actin) assemblies at cytomembrane zones and change the kinetic patterns of vesicle exocytosis. Our finding shapes the first quantitative picture of neurotoxicity induced by high-concentration nanoplastics exposure at a single-cell level.

Highly Stable and Selective Sensing of Hydrogen Sulfide in Living Mouse Brain with NiN4 Single-Atom Catalyst-Based Galvanic Redox Potentiometry.

Hydrogen sulfide (H2S) is recognized as a gasotransmitter and multifunctional signaling molecule in the central nervous system. Despite its essential neurofunctions, the chemical dynamics of H2S during physiological and pathological processes remains poorly understood, emphasizing the significance of H2S sensor development. However, the broadly utilized electrochemical H2S sensors suffer from low stability and sensitivity loss in vivo due to sulfur poisoning-caused electrode passivation. Herein, we report a high-performance H2S sensor that combines single-atom catalyst strategy and galvanic redox potentiometry to overcome the issue. Atomically dispersed NiN4 active sites on the sensing interface promote electrochemical H2S oxidation at an extremely low potential to drive spontaneous bipolarization of a single carbon fiber. Bias-free potentiometric sensing at open-circuit condition minimizes sulfur accumulation on the electrode surface, thus significantly enhancing the stability and sensitivity. The resulting sensor displays high selectivity to H2S against physiological interferents and enables real-time accurate quantification of H2S-releasing behavior in the living mouse brain.

[Analytical method of organophosphorus flame retardants in fish by ultra-high performance liquid chromatography-tandem mass spectrometry based on EMR-Lipid purification].

OBJECTIVE: A method for simultaneous determination of 11 kinds of organophosphorus flame retardants in fish was established by using the EMR-Lipid cleaning agent and ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). METHODS: The samples were extracted by ultrasonic with 0.5% formic acid acetonitrile solution. The lipid removal product EMR-lipid was used for lipid purification. The co-extracts were further removed by magnesium sulfate, N-propyl ethylene amine(PSA) and graphitized carbon black(GCB) purification agent. The target compounds were separated on an ACQUITY UPLC® BEH C_(18) column(100 mm×2.1 mm, 1.7 µm), detected by electrospray ionization(ESI) and positive ion multiple reaction monitoring mode, the internal standard method and external standard method are quantitatively evaluated, and external standard method was adopted. RESULTS: The 11 kinds of organophosphorus flame retardants had a good linear relationship in the range of 0.5-50 µg/L(tris(2-ethylhexyl) phosphate 0.05-5 µg/L) with r>0.999. The limits of detection were 0.004-1.029 µg/kg and the limits of quantitation were 0.012-3.094 µg/kg. The average recoveries at three spiked levels(low, medium and high) were 80.0%-111.2% with the relative standard deviations all less than 10%(n=6). CONCLUSION: The method could be used for the determination of trace organophosphorus flame retardants in freshwater fish with accurate and reliable result.

Synergistic Charge Percolation in Conducting Polymers Enables High-Performance In Vivo Sensing of Neurochemical and Neuroelectrical Signals.

Challenges remain in establishing a universal method to precisely tune electrochemical properties of conducting polymers for multifunctional neurosensing with high selectivity and sensitivity. Here, we demonstrate a facile and general approach to achieving synergistic charge percolation in conducting polymers (i.e., poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), PEDOT:PSS) by incorporating conductive catalysts (i.e., carbon nanotubes, CNTs) and post-processing. The approach shows synergistic effects: (i) CNTs and post-processing together promote PEDOT ordered interconnection for highly efficient charge percolation that accelerates electrochemical kinetics; (ii) CNTs catalyze the electrooxidation of vitamin C for selective electrochemical sensing; (iii) CNTs enhance the charge storage/injection capacity of PEDOT:PSS. The prepared CNT-PEDOT:PSS fiber mechanically matches with neural tissues and is proved to be a biocompatible versatile microsensor capable of high-performance neurosensing in vivo.

Vitamin D Inhibits the Early Aggregation of α-Synuclein and Modulates Exocytosis Revealed by Electrochemical Measurements.

Alpha-synuclein (α-Syn) localizes at presynaptic terminal and modulates synaptic functions. Increasing evidence demonstrate that α-Syn oligomers, forming at the early of aggregation, are cytotoxic and is thus related to brain neurodegenerative diseases. Herein, we find that vitamin D (VD) can reduce neurocytotoxicity. The reduced neurocytotoxicity might be attributed to the less amount of large-sized α-Syn oligomers inhibited by VD, measured by electrochemical collision at single particle level, which are not observable with traditionally ensembled method. Single-cell amperometry (SCA) results show that VD can recover the amount of neurotransmitter release during exocytosis induced by α-Syn oligomers, further verifying the neuroprotection of VD. Our study reveals the neuroprotective role of VD through inhibiting α-Syn aggregation, which is envisioned to be of great importance in treatment and prevention of the neurodegenerative diseases.

Support-Free PEDOT:PSS Fibers as Multifunctional Microelectrodes for In Vivo Neural Recording and Modulation.

In vivo microelectrodes are essential for neuroscience studies. However, development of microelectrodes with both flexibility and multifunctionality for recording chemical and electrical signals in the same extracellular microspace and modulating neural activity remains challenging. Here, we find that pure PEDOT:PSS fibers (i.e., support-free) exhibit high conductivity, fast heterogeneous electron transfer, and suitable charge storage and injection capabilities, and can thus directly act as microelectrodes not only for chemical and electrophysiological recording in the same extracellular microspace, but also for electromodulation of neural microcircuit activity. Moreover, the microelectrodes mechanically match with neural tissues, exhibiting less foreign body responses. Given the multifunctionality, flexibility, and biocompatibility, the support-free PEDOT:PSS-based microelectrodes offer a new avenue to microelectrode technology for neuroscience research, diagnostics and therapeutics.

Ag2S/Ag Nanoparticle Microelectrodes for In Vivo Potentiometric Measurement of Hydrogen Sulfide Dynamics in the Rat Brain.

Hydrogen sulfide (H2S) plays a pivotal role in gas signal transduction, neuroprotection, and regulation of physiological and pathological processes. However, in vivo tracking the dynamic of hydrogen sulfide in the complex brain environment still faces huge challenges. This study demonstrates a new potentiometric method to monitor in vivo the dynamics of hydrogen sulfide in the rat brain using silver nanoparticles (AgNPs)-modified carbon fiber microelectrodes (AgNPs/CFE) pretreated with Na2S (i.e., Ag2S/AgNPs/CFE), which acts as a solid-contact and ion-selective microelectrode. The Ag2S/AgNPs/CFE exhibits good potential response toward hydrogen sulfide in the range of 2.5-160 µM, with a detection limit of 0.8 µM. Because of the presence of Ag2S, the Ag2S/AgNPs/CFE shows good selectivity to hydrogen sulfide, avoiding the interference from coexistent electroactive neurochemicals and the analogies, such as ascorbic acid and cysteine in the central nervous system. This good selectivity combined with the reversibility, protein antifouling, and biocompatibility of the microelectrode enables the Ag2S/AgNPs/CFE to detect hydrogen sulfide in the rat brain during local microinfusion of Na2S and the change in pH. Our study provides a reliable method to track hydrogen sulfide selectively in vivo, which will help to explore the function of hydrogen sulfide in neurophysiology and pathology.

Simultaneous Monitoring of Intracellular Temperature and Norepinephrine Variation by Fluorescent Probes during Norepinephrine Reuptake.

A long-standing challenge has been the simultaneous sensing of intracellular temperature and norepinephrine (NE) variations to explore signaling pathways and depression pathogeny. Here, we designed a fluorescent probe using poly(N-isopropylacrylamide) and 1-[4-(7-nitro-benzo [1,2,5]oxadiazol-4-yl)-piperazin-1-yl]-propenone (PNIPAm-AANBD) and (E)-1-(4-boronobenzyl)-2-(2-(1,3-dioxo-1H,3H-benzo[de]isochromen-6-yl)vinyl)pyridin-1-ium bromide (PHE) for simultaneously measuring the temperature and NE with high selectivity. The fluorescence intensity of the PNIPAm-AANBD moiety exhibited a good response to temperature changes. The PHE moiety could selectively sense NE due to the naphthalic anhydride group in PHE, which formed naphthalimide upon bonding with the primary amino group of NE. The hydroxyl-terminated ligand recognized the phenolic hydroxyl group of NE through the formation of hydrogen bonds. Using the proposed fluorescent probe, variations in the intracellular temperature and NE during NE reuptake could be simultaneously measured. It was first discovered that with the inhibition of antidepressant drugs, the intracellular temperature increased by 1.2-2.1 °C, and the NE reuptake decreased by about 21.5 µM. The measured variations in intracellular temperature and NE during neurotransmitter reuptake can shed light on the underlying mechanism of neurotransmitter signaling pathways, which may facilitate the treatment of depression.

[Simultaneous determination of 13 antibiotics in disinfection products by ultra-high performance liquid chromatography-tandem mass spectrometry].

OBEJECTIVE: To develop a method for the determination of 13 antibiotics in 8 classes for desinfection products by ulta-high perfomance chromatography-tandem mass spectrometry(UPLC-MS/MS). METHODS: Samples were extracted by methanol or acetonitrile. The target compouds were separated on a Waters HSS T3 column(100 mm×2. 1 mm, 1. 8 µm), and detected by triple quadrupole tandem mass spectrometer. RESULTS: The 13 selected antibiotics showed good linear relationships in the range of 4-100 µg/L and the correlation coefficients(r~2) were all above 0. 991. The limits of detection ranged from 2 to 25 µg/kg. The recovery rates at three spiked levels(low, medium and high) in three dosage forms of disinfection products were in the range of 71. 2%-130. 4%, and the relative standard deviations(RSD) were all less than 11. 3%, which could meet the detection requirements of illegal addition of antibiotics in disinfection products. Ofloxacin at a concentration of 21. 1 mg/kg was found in a cream disinfection product by the developed method, and no related drugs were detected in other samples. CONCLUSION: This method is simple, reliable, reproducible, which covers a wide range of antibiotics, and provides technical support for monitoring the illegal addition of antibiotics in disinfection products.

Deep Learning for Voltammetric Sensing in a Living Animal Brain.

Numerous neurochemicals have been implicated in the modulation of brain function, making them appealing analytes for sensors and diagnostics. However, it is a grand challenge to selectively measure multiple neurochemicals simultaneously in vivo because of their great variations in concentrations, dynamic nature, and composition. Herein, we present a deep learning-based voltammetric sensing platform for the highly selective and simultaneous analysis of three neurochemicals in a living animal brain. The system features a carbon fiber electrode capable of capturing the mixed dynamics of a neurotransmitter, neuromodulator, and ions. Then a powerful deep neural network is employed to resolve individual chemical and spatial-temporal information. With this, a single electrochemical measurement reveals an interplaying concentration changes of dopamine, ascorbate, and ions in living rat brain, which is unobtainable with existing analytical methodologies. Our strategy provides a powerful means to expedite research in neuroscience and empower sensing-aided diagnostic applications.

Electrochemically Probing Dynamics of Ascorbate during Cytotoxic Edema in Living Rat Brain.

Cytotoxic edema is the initial and most important step in the sequence that almost inevitably leads to brain damage. Exploring the neurochemical disturbances in this process is of great significance in providing a measurable biological parameter for signaling specific pathological conditions. Here, we present an electrochemical system that pinpoints a critical neurochemical involved in cytotoxic edema. Specially, we report a molecularly tailored brain-implantable ascorbate sensor (CFEAA2.0) featuring excellent selectivity and spatiotemporal resolution that assists the first observation of release of ascorbate induced by cytotoxic edema in vivo. Importantly, we reveal that this release is associated with an increase in the amount of cytotoxic edema-inducing agent and that blockage of cytotoxic edema abolishes ascorbate release, further supporting that ascorbate efflux is cytotoxic edema-dependent. Our study holds the promise for understanding the molecular basis of cytotoxic edema that can lead to the discovery of biomarkers or potential therapeutic strategies of brain diseases.

Single-Atom Co-N4 Electrocatalyst Enabling Four-Electron Oxygen Reduction with Enhanced Hydrogen Peroxide Tolerance for Selective Sensing.

Electrocatalysis of the four-electron oxygen reduction reaction (ORR) provides a promising approach for energy conversion, storage, and oxygen monitoring. However, it is always accompanied by the reduction of hydrogen peroxide (H2O2) on most employed catalysts, which brings down the electrocatalytic selectivity. Here, we report a single-atom Co-N4 electrocatalyst for the four-electron ORR at an onset potential of 0.68 V (vs RHE) in neutral media while with high H2O2 tolerance, outperforming commercial Pt electrocatalysts. Electrochemical kinetic analysis confirms that the Co-N4 catalytic sites dominantly promote the direct four-electron pathway of the ORR rather than the two sequential two-electron reduction pathways with H2O2 as the intermediate. Density functional theory calculations reveal that H2O2 reduction is hampered by the weak adsorption of H2O2 on the porphyrin-like Co centers. This endows the electrocatalyst with improved resistance to current interference from H2O2, enabling highly selective O2 sensing as validated by the reliable sensing performance in vivo. Our study demonstrates the intriguing advantage of single-atom catalysts with high capacity for tailoring metal-adsorbate interactions, broadening their applications in environmental and life monitoring.