Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry

We applied electrochemical techniques with nano‐tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 μm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc‐treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.     

Dynamic Gas‐Inclusion in a Single Crystal

In solid‐state science, most changing phenomena have been mysterious. Furthermore, the changes in chemical composition should be added to mere physical changes to also cover the chemical changes. Here, the first success in characterizing the nature of gas inclusion in a single crystal is reported. The gas inclusion process has been thoroughly investigated by in situ optical microscopy, single‐crystal X‐ray diffraction analyses, and gas adsorption measurements. The results demonstrated an inclusion action of a first‐order transition behavior induced by a critical concentration on the phase boundary. The transfer of phase boundary and included gas are strongly related. This relationship can generate the dynamic features hidden in the inclusion phenomena, which can lead to the guest capturing and transfer mechanism that can apply to spatiotemporal inclusion applications by using host solids.     

硫脲氧化反应动力学研究进展

本文综述了硫脲氧化反应动力学的研究进展,根据氧化剂和氧化方式不同,将硫脲氧化体系分成含卤氧化体系和非卤氧化体系两大类,其中含卤氧化体系包括亚氯酸、碘酸、溴酸、卤素单质氧化硫脲的反应体系;非卤氧化体系包括双氧水、自由基、电化学和金属酸盐氧化硫脲的反应体系。总结了不同反应体系的动力学现象和反应机理研究状况,文中还介绍了在硫脲氧化反应动力学研究中光电磁及色谱方法的发展状况,提出硫脲氧化反应动力学机理研究突破可能途径。     

Redox‐Filled Carbon‐Fiber Microelectrodes for Single‐Cell Exocytosis

Carbon‐fiber microelectrodes (CFEs) are the primary electroanalytical tool in single‐cell exocytosis and in vivo studies. Here we report a new study on the kinetic properties of electrolyte‐filled CFEs in single‐cell measurements and demonstrate that the addition of outer sphere redox species, such as Fe(CN)₆^3? and Ru(NH₃)₆³⁺, in the backfill electrolyte solution can greatly enhance the kinetic response of CFEs. We show that at 750 mV, a voltage normally applied for detection of dopamine, the presence of fast outer sphere redox species in the backfilling solution significantly enhances the kinetic response of CFEs toward fast dopamine detection at single PC12 cells. Moreover, we also demonstrate that the use of Fe(CN)₆^3? in the backfilling solution has enabled direct measurement of dopamine at applied voltages as low as 200 mV. This kinetic enhancement is believed to be due to faster electron‐transfer kinetics on the coupling pole as compared to the sluggish reduction of oxygen. We anticipate that such redox‐filled CFE ultramicroelectrodes will find many useful applications in single cell exocytosis and in vivo sensing.     

Probing Second Harmonic Components of pH‐Sensitive Redox Processes in a Mesoporous TiO2‐Nafion Film Electrode with Fourier‐Transformed Large‐Amplitude Sinusoidally Modulated Voltammetry

Electrochemical processes in mesoporous TiO₂‐Nafion thin films deposited on indium tin oxide (ITO) electrodes are inherently complex and affected by capacitance, Ohmic iR‐drop, RC‐time constant phenomena, and by potential and pH‐dependent conductivity. In this study, large‐amplitude sinusoidally modulated voltammetry (LASMV) is employed to provide access to almost purely Faradaic‐based current data from second harmonic components, as well as capacitance and potential domain information from the fundamental harmonic for mesoporous TiO₂‐Nafion film electrodes. The LASMV response has been investigated with and without an immobilized one‐electron redox system, ferrocenylmethyltrimethylammonium⁺. Results clearly demonstrate that the electron transfer associated with the immobilized ferrocene derivative follows two independent pathways i) electron hopping within the Nafion network and ii) conduction through the TiO₂ backbone. The pH effect on the voltammetric response for the TiO₂ reduction pathway (ii) can be clearly identified in the 2^nd harmonic LASMV response with the diffusion controlled ferrocene response (i) acting as a pH independent reference. Application of second harmonic data derived from LASMV measurement, because of the minimal contribution from capacitance currents, may lead to reference‐free pH sensing with systems like that found for ferrocene derivatives.     

Barcoding Biological Reactions with DNA‐Functionalized Vesicles

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase‐segregated membranes, promote fusion between specific vesicle populations. Membrane phase‐segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA‐mediated fusion events. The orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. Vesicle fusion between DNA‐tethered vesicles can be used to initiate in vitro protein expression to produce model soluble and membrane proteins. Engineering orthogonal fusion events between DNA‐tethered vesicles provides a new strategy to control the spatiotemporal dynamics of cell‐free reactions, expanding opportunities to engineer artificial cellular systems.     

RGB‐Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca²⁺ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.     

Synthesis of 1‐amino‐2‐methylindoline by Raschig process: Kinetics of the oxidation of 1‐amino‐2‐methylindoline by chloramine

The synthesis of 1‐amino‐2‐methylindoline by the Raschig process was undertaken in aqueous solution. The principal side reaction that occurs in the medium is the oxidation of 1‐amino‐2‐methylindoline formed by chloramine. To increase the yield of 1‐amino‐2‐methylindoline, its oxidation by chloramine was studied by GC and HPLC at various concentrations of reactants and for a pH interval ranging between 9.9 and 13.5. The reaction is bimolecular and exhibits a specific acid catalysis. In alkaline medium, 1‐amino‐2‐methylindole is the principal product. The enthalpy and entropy of activation were determined at pH 12.89. In unbuffered solution, the interaction was autocatalyzed by the ammonium ions formed, which indicates a competitive oxidation of neutral and ionic forms of 1‐amino‐2‐methylindoline by chloramine. A mathematical treatment based on one implicit equation allows a quantitative interpretation of all the phenomena observed over the above pH interval. It takes both acid–base dissociation equilibrium and alkaline hydrolysis of chloramine into account. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 515–523, 2002     

Tunable and Photoswitchable Chemically Induced Dimerization for Chemo‐optogenetic Control of Protein and Organelle Positioning

The spatiotemporal dynamics of proteins and organelles play an important role in controlling diverse cellular processes. Optogenetic tools using photosensitive proteins and chemically induced dimerization (CID), which allow control of protein dimerization, have been used to elucidate the dynamics of biological systems and to dissect the complicated biological regulatory networks. However, the inherent limitations of current optogenetic and CID systems remain a significant challenge for the fine‐tuning of cellular activity at precise times and locations. Herein, we present a novel chemo‐optogenetic approach, photoswitchable chemically induced dimerization (psCID), for controlling cellular function by using blue light in a rapid and reversible manner. Moreover, psCID is tunable; that is, the dimerization and dedimerization degrees can be fine‐tuned by applying different doses of illumination. Using this approach, we control the localization of proteins and positioning of organelles in live cells with high spatial (μm) and temporal (ms) precision.     

pH‐Responsive Drug‐Delivery Systems

In many biomedical applications, drugs need to be delivered in response to the pH value in the body. In fact, it is desirable if the drugs can be administered in a controlled manner that precisely matches physiological needs at targeted sites and at predetermined release rates for predefined periods of time. Different organs, tissues, and cellular compartments have different pH values, which makes the pH value a suitable stimulus for controlled drug release. pH‐Responsive drug‐delivery systems have attracted more and more interest as “smart” drug‐delivery systems for overcoming the shortcomings of conventional drug formulations because they are able to deliver drugs in a controlled manner at a specific site and time, which results in high therapeutic efficacy. This focus review is not intended to offer a comprehensive review on the research devoted to pH‐responsive drug‐delivery systems; instead, it presents some recent progress obtained for pH‐responsive drug‐delivery systems and future perspectives. There are a large number of publications available on this topic, but only a selection of examples will be discussed.     

Tunable Visible and Near‐IR Photoactivation of Light‐Responsive Compounds by Using Fluorophores as Light‐Capturing Antennas

Although the corrin ring of vitamin B₁₂ is unable to efficiently absorb light beyond 550 nm, it is shown that commercially available fluorophores can be used as antennas to capture long‐wavelength light to promote scission of the Co? C bond at wavelengths up to 800 nm. The ability to control the molecular properties of bioactive species with long visible and near‐IR light has implications for drug delivery, nanotechnology, and the spatiotemporal control of cellular behavior.     

pH‐Induced Shape‐Memory Polymers

A novel pH sensitive shape‐memory polymer (SMP) is prepared by cross‐linking the β‐cyclodextrin modified alginate (β‐CD‐Alg) and diethylenetriamine modified alginate (DETA‐Alg): The pH reversible β‐CD‐DETA inclusion complexes serve as a reversible phase, and the cross‐linked alginate chains serve as a fixing phase. It is shown that this material can be processed into temporary shape as we needs at pH 11.5 and recover to its initial shape at pH 7. The recovery ratio and the fixity ratio were 95.7 ± 0.9% and 94.8 ± 1.1%, respectively. Furthermore, this material showed good degradability and biocompatibility. Because the shape transition pH value is quite close to that of our body fluid and this pH triggered shape‐memory effect is convenient and safe to use, this material has a high potential for medical application.     

Live‐Cell Imaging of Endogenous mRNAs with a Small Molecule

Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non‐engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene‐specific RNA aptamer, combined with a cell‐permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live‐cell imaging of the endogenous mRNA of β‐actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin‐2, cortactin, and cytoplasmic FMR1‐interacting protein 2. The RNA‐imaging technology and its further optimization might permit live‐cell imaging of any RNA molecules.     

High‐Throughput Development of a Hybrid‐Type Fluorescent Glutamate Sensor for Analysis of Synaptic Transmission

Fluorescent sensors are powerful tools for visualizing cellular molecular dynamics. We present a high‐throughput screening system, designated hybrid‐type fluorescence indicator development (HyFInD), to identify optimal position‐specific fluorophore labeling in hybrid‐type sensors consisting of combinations of ligand‐binding protein mutants with small molecular fluorophores. We screened sensors for glutamate among hybrid molecules obtained by the reaction of four cysteine‐reactive fluorescence probes with a set of cysteine‐scanning mutants of the 274 amino acid S1S2 domain of AMPA‐type glutamate receptor GluA2 subunit. HyFInD identified a glutamate‐responsive probe (enhanced glutamate optical sensor: eEOS) with a dynamic range >2400 %, good photostability, and high selectivity. When eEOS was specifically tethered to neuronal surfaces, it reliably visualized the spatiotemporal dynamics of glutamate release at single synapses, revealing synapse‐to‐synapse heterogeneity of short‐term plasticity.     

Biocatalytic Feedback‐Driven Temporal Programming of Self‐Regulating Peptide Hydrogels

Switchable self‐assemblies respond to external stimuli with a transition between near‐equilibrium states. Although being a key to present‐day advanced materials, these systems respond rather passively, and do not display autonomous dynamics. For autonomous behavior, approaches must be found to orchestrate the time domain of self‐assemblies, which would lead to new generations of dynamic and self‐regulating materials. Herein, we demonstrate catalytic control of the time domain of pH‐responsive peptide hydrogelators in a closed system. We program transient acidic pH states by combining a fast acidic activator with the slow, enzymatic, feedback‐driven generation of a base (dormant deactivator). This transient state can be programmed over orders of magnitude in time. It is coupled to dipeptides to create autonomously self‐regulating, dynamic gels with programmed lifetimes, which are used for fluidic guidance, burst release, and self‐erasing rapid prototyping.     

pH‐induced structural transformation of N,N′‐diaspartic acid‐3, 4, 9, 10‐tetracarboxylic diimide as observed by scanning probe microscopy

To investigate the acidity of aspartic acid, the N, N′‐diasparitic acid‐3, 4, 9, 10‐perylene tetracarboxylic diimide (NAAPD) was synthesized and characterized. Previous studies have examined the self‐assembly behaviours of NAAPD mainly using atomic force microscopy at various pH levels. The present study sought to examine the characteristics and dynamics of NAAPD assembly using scanning probe microscopy. Our experimental results suggest that the assembly of NAAPD nanostructures may be regulated by pH. This phenomenon may be attributed to the deprotonation and properties of the carboxyl groups within aspartic acid, which is consistent with the interactions revealed from studies using scanning tunnelling microscopy. Copyright © 2016 John Wiley & Sons, Ltd.     

Surface pressure‐area curves compression isotherm of sericin Langmuir monolayer

The Langmuir monolayer of sericin protein was studied by means of surface pressure (π)—molecular area (A) isotherms at different pH subphase. The monolayer of sericin exhibits typical phase transition phenomena at pH 2, pH 4.8, pH 7 and pH 11, respectively, including from gas state to gas‐liquid state and finally to condensed solid state. However, the monolayer of sericin on pH 11 subphase appears to be solid state. Copyright © 2007 John Wiley & Sons, Ltd.     

Multidirectional Activity Control of Cellular Processes by a Versatile Chemo‐optogenetic Approach

The spatiotemporal dynamics of proteins or organelles plays a vital role in controlling diverse cellular processes. However, acute control of activity at distinct locations within a cell is challenging. A versatile multidirectional activity control (MAC) approach is presented, which employs a photoactivatable system that may be dimerized upon chemical inducement. The system comprises second‐generation SLF*‐TMP (S*T) and photocaged NvocTMP‐Cl dimerizers; where, SLF*‐TMP features a synthetic ligand of the FKBP(F36V) binding protein, Nvoc is a caging group, and TMP is the antibiotic trimethoprim. Two MAC strategies are demonstrated to spatiotemporally control cellular signaling and intracellular cargo transport. The novel platform enables tunable, reversible, and rapid control of activity at multiple compartments in living cells.     

Effects of solute-solvent proton exchange on polypeptide chain dynamics: a constant-pH molecular dynamics study

A method for performing implicit-solvent molecular dynamics simulations at constant pH was applied to a pentapeptide acetyl-Ala-Asp-Ala-Lys-Ala-amide at pH 4. As a reference, molecular dynamics simulations were done for the same peptide with two variants of its fixed protonation patterns expected to dominate at pH 4, i.e., with a protonated and a deprotonated side chain of the Asp residue and the protonated Lys residue in both cases. The dynamic trajectories of the peptide were used to discuss the problem of the significance of the solute-solvent proton exchange phenomena for the dynamics and structural distributions of the polypeptide chain. The Asp-Lys distance was used as a probe of the overall molecular structure of the investigated pentapeptide. To characterize the dynamics, distributions of the "waiting" times for a transition from a "short" distance conformation to a "long" distance conformation were constructed, based on the generated molecular dynamics trajectories. We show that the relaxation time for the transitions, derived from the constant-pH simulations, is very close to the relaxation time characterizing a permanently protonated molecule, although the average protonation probability of the short-distance conformation is close to zero. However, the distribution of the Asp-Lys distances obtained from constant-pH simulations cannot be reproduced as a linear combination of the distributions resulting from the simulations with fixed protonation states.     

Spatiotemporal Dynamics of Aggregation‐Induced Emission Enhancement Controlled by Optical Manipulation

We present spatiotemporal control of aggregation‐induced emission enhancement (AIEE) of a protonated tetraphenylethene derivative by optical manipulation. A single submicrometer‐sized aggregate is initially confined by laser irradiation when its fluorescence is hardly detectable. The continuous irradiation of the formed aggregate leads to sudden and rapid growth, resulting in bright yellow fluorescence emission. The fluorescence intensity at the peak wavelength of 540 nm is tremendously enhanced with growth, meaning that AIEE is activated by optical manipulation. Amazingly, the switching on/off of the activation of AIEE is arbitrarily controlled by alternating the laser power. This result means that optical manipulation increases the local concentration, which overcomes the electrostatic repulsion between the protonated molecules, namely, optical manipulation changes the aggregate structure. The dynamics and mechanism in AIEE controlled by optical manipulation will be discussed from the viewpoint of molecular conformation and association depending on the laser power.