Strong correlations and Fickian water diffusion in narrow carbon nanotubes

The authors have used atomistic molecular dynamics (MD) simulations to study the structure and dynamics of water molecules inside an open ended carbon nanotube placed in a bath of water molecules. The size of the nanotube allows only a single file of water molecules inside the nanotube. The water molecules inside the nanotube show solidlike ordering at room temperature, which they quantify by calculating the pair correlation function. It is shown that even for the longest observation times, the mode of diffusion of the water molecules inside the nanotube is Fickian and not subdiffusive. They also propose a one-dimensional random walk model for the diffusion of the water molecules inside the nanotube. They find good agreement between the mean-square displacements calculated from the random walk model and from MD simulations, thereby confirming that the water molecules undergo normal mode diffusion inside the nanotube. They attribute this behavior to strong positional correlations that cause all the water molecules inside the nanotube to move collectively as a single object. The average residence time of the water molecules inside the nanotube is shown to scale quadratically with the nanotube length.     

用于检测生物体系中线粒体活性氧的荧光探针

In addition to being the energy powerhouse of the cell, mitochondria are an important source of reactive oxygen species (ROS) during the process of molecular oxygen metabolism. Mitochondrial ROS are closely associated with normal physiological functions as well as human diseases, and participate in cell signaling, nucleic acid and protein damage, and oxidative stress induction. However, the complicated interplay between mitochondrial ROS and the cellular pathological state has not been fully elucidated. It is expected that research on the mitochondrial ROS undertaking in the molecular pathogenesis of human diseases would benefit from development of efficient tools for the detection of these ROS. In recent years, an increasing number of fluorescent probes for mitochondrial ROS with high sensitivity and selectivity have been developed. Here, we present a review of the recent advances in small molecular fluorescent probes for selective detection of ROS inside the mitochondria. In this review, the design, synthesis, characteristics, and applications of the published fluorescent probes for mitochondrial ROS are discussed in detail.     

Cell-Penetrating Mitochondrion-Targeting Ligands for the Universal Delivery of Small Molecules,Proteins and Nanomaterials

Mitochondria are key organelles that perform vital cellular functions such as those related to cell survival and death. The targeted delivery of different types of cargos to mitochondria is a well-established strategy to study mitochondrial biology and diseases. Of the various existing mitochondrion-transporting vehicles, most suffer from poor cytosolic entry, low delivery efficiency, limited cargo types, and cumbersome preparation protocols, and none was known to be universally applicable for mitochondrial delivery of different types of cargos (small molecules, proteins, and nanomaterials). Herein, two new cell-penetrating, mitochondrion-targeting ligands (named Mito^Ligand) that are capable of effectively “tagging” small-molecule drugs, native proteins and nanomaterials are disclosed, as well as their corresponding chemoselective conjugation chemistry. Upon successful cellular delivery and rapid endosome escape, the released native cargos were found to be predominantly localized inside mitochondria. Finally, by successfully delivering doxorubicin, a well-known anticancer drug, to the mitochondria of HeLa cells, we showed that the released drug possessed potent cell cytotoxicity, disrupted the mitochondrial membrane potential and finally led to apoptosis. Our strategy thus paves the way for future mitochondrion-targeted therapy with a variety of biologically active agents.     

Real‐Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core–Shell Nanowire Electrodes

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living‐cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC‐core–shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano‐core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles.     

The behavior of reorientational correlation functions of water at the water-lipid bilayer interface

We studied the effects of confinement and the head group motion on the behavior of the reorientational correlation functions for water molecules at the water/lipid bilayer interface. The correlation functions were calculated from the data obtained from two molecular dynamics simulations: one with a flexible bilayer and the other with a frozen bilayer. In our present analysis the water molecules were separated into spatial regions according to their distance from the bilayer surface and into population groups, according to the length of their stay in the corresponding regions. We estimate that for most of the water molecules that are in a strongly confined environment of the transition region between the head groups and tails, and that solvate carbonyl groups, the decay time of their reorientational correlation functions is of the order of a few tens of picoseconds. Water molecules that stay inside the transition region for long periods of time can display longer time decay (of the order of hundreds of picoseconds). This latter long time decay is determined by the dynamics of the phospholipids, it is substantially reduced when the bilayer is frozen. The decay of the correlation functions for the interfacial water molecules that are solvating the head groups is also slowed down when compared to bulk, but just by factors of 3-4.     

Metal Complexes or Chelators with ROS Regulation Capacity: Promising Candidates for Cancer Treatment

Reactive oxygen species (ROS) are rapidly eliminated and reproduced in organisms, and they always play important roles in various biological functions and abnormal pathological processes. Evaluated ROS have frequently been observed in various cancers to activate multiple pro-tumorigenic signaling pathways and induce the survival and proliferation of cancer cells. Hydrogen peroxide (H₂O₂) and superoxide anion (O₂^•−) are the most important redox signaling agents in cancer cells, the homeostasis of which is maintained by dozens of growth factors, cytokines, and antioxidant enzymes. Therefore, antioxidant enzymes tend to have higher activity levels to maintain the homeostasis of ROS in cancer cells. Effective intervention in the ROS homeostasis of cancer cells by chelating agents or metal complexes has already developed into an important anti-cancer strategy. We can inhibit the activity of antioxidant enzymes using chelators or metal complexes; on the other hand, we can also use metal complexes to directly regulate the level of ROS in cancer cells via mitochondria. In this review, metal complexes or chelators with ROS regulation capacity and with anti-cancer applications are collectively and comprehensively analyzed, which is beneficial for the development of the next generation of inorganic anti-cancer drugs based on ROS regulation. We expect that this review will provide a new perspective to develop novel inorganic reagents for killing cancer cells and, further, as candidates or clinical drugs.     

Structure, dynamics, and the free energy of solute adsorption at liquid-vapor interfaces of simple dipolar systems: molecular dynamics results for pure and mixed Stockmayer fluids

We have performed molecular dynamics simulation studies of the structural, thermodynamic, and dynamical properties of liquid-vapor interfaces of pure and binary Stockmayer fluids of different polarity. The density profiles, the width of the liquid-vapor interface, and the orientational structure of the interfaces are calculated to characterize the structural aspects of the interfaces. Among the thermodynamic properties, we have computed the surface tension and also the free energy of transfer of a charged solute across the liquid-vapor interface for both pure and mixed fluids. Among the dynamical properties of the interfaces, we have calculated the time dependence of the velocity and angular velocity autocorrelation functions, continuous and intermittent survival probabilities, mean square displacements, diffusion coefficients, and also the dipole correlation functions and orientational relaxation times of interfacial solvent molecules. It is found that the width of the interfaces decreases with increase of concentration of the more polar component. The dipole vectors of the interfacial molecules tend to align parallel to the surfaces and this alignment is enhanced with increasing dipole moment of the fluid molecules. Also, the surface tension shows an increasing trend with increase of dipole moment of the molecules. The dynamical properties of the interfaces are found to be different from those of the corresponding bulk liquid phases. In general, the molecules at the interfaces are found to rotate and translate in the parallel direction at a somewhat faster rate than the bulk molecules. Also, on increase of concentration of the more polar component, the diffusion and orientational relaxation of interfacial molecules are found to show a weaker slowing down than those of the bulk molecules, which can be attributed to the preferential presence of the more polar component in the bulk liquid regions. The temporal behavior of the interfacial survival probabilities reveals a decrease of the survival times with increasing polarity, which can be attributed to a corresponding decrease in the interfacial thickness. Results are presented for both continuous and intermittent survival times and the origins of their differences are discussed. The free energy calculations reveal no minimum at the interfaces for adsorption of a charged solute, which shows that the ions would prefer to stay in the interior of the liquid phases, rather than at interfaces, for these model dipolar systems.     

Dynamics of water confined in the interdomain region of a multidomain protein

Molecular dynamics simulations are performed to study the dynamics of interfacial water confined in the interdomain region of a two-domain protein, BphC enzyme. The results show that near the protein surface the water diffusion constant is much smaller and the water-water hydrogen bond lifetime is much longer than that in bulk. The diffusion constant and hydrogen bond lifetime can vary by a factor of as much as 2 in going from the region near the hydrophobic domain surface to the bulk. Water molecules in the first solvation shell persist for a much longer time near local concave sites than near convex sites. Also, the water layer survival correlation time shows that on average water molecules near the extended hydrophilic surfaces have longer residence times than those near hydrophobic surfaces. These results indicate that local surface curvature and hydrophobicity have a significant influence on water dynamics.     

Optimisation of mould surface temperature and bottle residence time in mould for the carbonated soft drink PET containers

Polyethylene terephthalate (PET) bottles, which are usually produced by injection stretch blow moulding (ISBM) are widely used for carbonated soft drinks (CSD) storage and transportation. Stretch rod movement, blow pressure, preform temperature profile, mould surface temperature and material properties are among the most important factors affecting the final product's quality in terms of the thickness distribution, burst pressure and top-load resistance of the bottles. However, the residence time of the blown bottle inside the mould is also an important factor affecting its final properties. Especially in PET bottle production for hot fillings, the residence time is a very important factor because the longer the residence time the better the crystalline structure of the PET. In this production, the lid section is desired to have a fully crystalline form so that it can withstand hot fluids. In this study, the aim was to optimise the mould surface temperature and the blown bottle's residence time inside the mould for 1 L soft drink PET bottle production based on the final properties using the ECHIP 7 design of experiment (DOE) program. The method employed through this program was a quadratic one. Optimum process parameters were determined by the response surface method (RSM) and the process settings ensuring maximum top-load, burst pressure, Tg and degree of crystallinity were regarded to be optimum. It was found that the optimum mould surface temperature and blown bottle residence time inside the mould were 10 °C and 20 s, respectively.     

Glycosides, depression and suicidal behaviour: the role of glycoside-linked proteins

Nowadays depression and suicide are two of the most important worldwide public health problems. Although their specific molecular mechanisms are still largely unknown, glycosides can play a fundamental role in their pathogenesis. These molecules act presumably through the up-regulation of plasticity-related proteins: probably they can have a presynaptic facilitatory effect, through the activation of several intracellular signaling pathways that include molecules like protein kinase A, Rap-1, cAMP, cADPR and G proteins. These proteins take part in a myriad of brain functions such as cell survival and synaptic plasticity. In depressed suicide victims, it has been found that their activity is strongly decreased, primarily in hippocampus and prefrontal cortex. These studies suggest that glycosides can regulate neuroprotection through Rap-1 and other molecules, and may play a crucial role in the pathophysiology of depression and suicide.     

Nanosecond to microsecond protein dynamics probed by magnetic relaxation dispersion of buried water molecules

Large-scale protein conformational motions on nanosecond-microsecond time scales are important for many biological processes, but remain largely unexplored because of methodological limitations. NMR relaxation methods can access these time scales if protein tumbling is prevented, but the isotropy required for high-resolution solution NMR is then lost. However, if the immobilized protein molecules are randomly oriented, the water 2H and 17O spins relax as in a solution of freely tumbling protein molecules, with the crucial difference that they now sample motions on all time scales up to approximately 100 micros. In particular, the exchange rates of internal water molecules can be determined directly from the 2H or 17O magnetic relaxation dispersion (MRD) profile. This possibility opens up a new window for characterizing the motions of individual internal water molecules as well as the large-scale protein conformational fluctuations that govern the exchange rates of structural water molecules. We introduce and validate this new NMR method by presenting and analyzing an extensive set of 2H and 17O MRD data from cross-linked gels of two model proteins: bovine pancreatic trypsin inhibitor and ubiquitin. We determine residence times and order parameters of four internal water molecules in these proteins and show that they are quantitatively consistent with the information available from crystallography and solution MRD. We also show how slow motions of side-chains bearing labile hydrogens can be monitored by the same approach. Proteins of any size can be studied at physiological hydration levels with this method.     

Investigation of dipolar-mediated water-protein interactions in microcrystalline Crh by solid-state NMR spectroscopy

Water-protein interactions play a major role in protein folding, structure, and function, and solid-state NMR has recently been shown to be a powerful tool for the site-resolved observation of these interactions in solid proteins. In this article we report investigations on possible water-protein dipolar transfer mechanisms in the microcrystalline deuterated protein Crh by a set of solid-state NMR techniques. Double-quantum (DQ) filtered and edited heteronuclear correlation experiments are used to follow direct dipolar water-protein magnetization transfers. Experimental data reveal no evidence for "solid-like" water molecules, indicating that residence times of solvent molecules are shorter than required for DQ creation, typically a few hundred microseconds. An alternative magnetization pathway, intermolecular cross-relaxation via heteronuclear nuclear Overhauser effects (NOEs), is probed by saturation transfer experiments. The significant additional enhancements observed when irradiating at the water frequency can possibly be attributed to direct heteronuclear water-protein NOEs; however, a contribution from relayed magnetization transfer via chemical exchange or proton-proton dipolar mechanisms cannot be excluded.     

Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions

Complexity and heterogeneity are common denominators of the many molecular events taking place inside the cell. Single-molecule techniques are important tools to quantify the actions of biomolecules. Heterogeneous interactions between multiple proteins, however, are difficult to study with these technologies. One solution is to integrate optical trapping with micro-fluidics and single-molecule fluorescence microscopy. This combination opens the possibility to study heterogeneous/complex protein interactions with unprecedented levels of precision and control. It is particularly powerful for the study of DNA-protein interactions as it allows manipulating the DNA while at the same time, individual proteins binding to it can be visualized. In this work, we aim to illustrate several published and unpublished key results employing the combination of fluorescence microscopy and optical tweezers. Examples are recent studies of the structural properties of DNA and DNA-protein complexes, the molecular mechanisms of nucleo-protein filament assembly on DNA and the motion of DNA-bound proteins. In addition, we present new results demonstrating that single, fluorescently labeled proteins bound to individual, optically trapped DNA molecules can already be tracked with localization accuracy in the sub-10 nm range at tensions above 1 pN. These experiments by us and others demonstrate the enormous potential of this combination of single-molecule techniques for the investigation of complex DNA-protein interactions.     

Solvation of p-coumaric acid in water

The photoactive yellow protein (PYP) is an important model protein for many (photoactive) signaling proteins. Key steps in the PYP photocycle are the isomerization and protonation of its chromophore, p-coumaric acid (pCA). In the ground state of the protein, this chromophore is in the trans configuration with its phenolic oxygen deprotonated. For this paper, we studied four different configurations of pCA solvated in water with ab initio molecular dynamics simulations as implemented in CP2K/Quickstep. We researched the influence of the protonation and isomerization state of pCA on its hydrogen-bonding properties and on the Mulliken charges of the atoms in the simulation. The chromophore isomerization state influenced the hydrogen-bonding less than its protonation state. In general, more charge yielded a higher hydrogen-bond coordination number. Where deprotonation increases both the coordination number and the residence time of the water molecules around the chromophore, protonation showed a somewhat lower coordination number on two of the three pCA oxygens but much higher residence times on all of them. This could be explained by the increased polarization of the OH groups of the molecule. The presence of the chromophore also influenced the charge and polarization of the water molecules around it. This effect was different in the four systems studied and mainly localized in the first solvation shell. We also performed a proton-transfer reaction from hydronium through various other water molecules to the chromophore. In this small charge-separated system, the protonation occurred within 6.5 ps. We identified the transition state for the final step in this protonation series.     

Interaction forces measured using AFM between colloids and surfaces coated with both dextran and protein

Both proteins and polysaccharides are biopolymers present on a bacterial surface that can simultaneously affect bacterial adhesion. To better understand how the combined presence of proteins and polysaccharides might influence bacterial attachment, adhesion forces were examined using atomic force microscopy (AFM) between colloids (COOH- or protein-coated) and polymer-coated surfaces (BSA, lysozyme, dextran, BSA+dextran and lysozyme+dextran) as a function of residence time and ionic strength. Protein and dextran were competitively covalently bonded onto glass surfaces, forming a coating that was 22-33% protein and 68-77% dextran. Topographic and phase images of polymer-coated surfaces obtained with tapping mode AFM indicated that proteins at short residence times (<1 s) were shielded by dextran. Adhesion forces measured between colloid and polymer-coated surfaces at short residence times increased in the order protein+dextran < or = protein < dextran. However, the adhesion forces for protein+dextran-coated surface substantially increased with longer residence times, producing the largest adhesion forces between polymer coated surfaces and the colloid over the longest residence times (50-100 s). It was speculated that with longer interaction times the proteins extended out from beneath the dextran and interacted with the colloid, leading to a molecular rearrangement that increased the overall adhesion force. These results show the importance of examining the effect of the combined adhesion force with two different types of biopolymers present and how the time of interaction affects the magnitude of the force obtained with two-polymer-coated surfaces.     

Two-site-hopping time correlation functions. Application to radical pair recombination

The two-site-hopping problem is studied in the time domain. The relaxation can be described as a product of two time correlation functions. One describes the time evolution for the average over the two sites, while the other accounts for the difference of the two sites. The latter exhibits an anomalous slow decay at long times and does not decay for fast hopping rates. The first does not depend on the hopping rate at all. The results are applied to the spin dynamics of novel pairwise-connected molecules carrying an unpaired electron spin.     

Single molecule dynamics on hydrophobic self-assembled monolayers

The interactions between adsorbate molecules and hydrophobic surfaces are of significant interest due to their importance in a variety of biological and separation processes. However, it is challenging to extrapolate macroscopic ensemble-averaged force measurements to molecular-level phenomena. Using total internal reflection fluorescence microscopy to image individual molecules at hydrophobic solid-aqueous interfaces, we directly observed dynamic behavior associated with the interactions between fluorescently labeled dodecanoic acid (our probe molecules) and self-assembled monolayers (SAM) comprising n-alkyltriethoxysilanes with systematically increasing chain length (from n = 4-18). In all cases, we observed at least two characteristic surface residence times and two diffusive modes, suggesting the presence of multiple distinct adsorbed populations. In general, the mean surface residence time increased and the mobility decreased with increasing SAM chain length, consistent with stronger probe-surface interactions. However, these trends were not primarily due to changes in characteristic residence times or diffusion coefficients associated with the individual populations but rather to a dramatic increase in the fraction associated with the long-lived slow-moving population(s) on long-chain SAMs. In particular, on longer (16-18 carbon) alkylsilane monolayers, the probe molecule exhibited far fewer desorption-mediated "flights" than on short (4-6 carbon) monolayers. Additionally, probes on the longer chain surfaces were much more likely to exhibit extended surface residence times as opposed to short transient surface visits.     

Quantifying the diffusion of a fluid through membranes by double phase encoded remote detection magnetic resonance imaging

We demonstrate that a position correlation magnetic resonance imaging (MRI) experiment based on two phase encoding steps separated by a delay can be used for quantifying diffusion across a membrane. This method is noninvasive, and no tracer substance or concentration gradient across the membrane is required. Because, in typical membranes, the T1 relaxation time of the fluid spins is usually much longer than the T2 time, we developed and implemented a new position correlation experiment based on a stimulated spin-echo, in which the relaxation attenuation of the signal is dominated by T1 instead of T2. This enables using relatively long delays needed in the diffusion measurements. The sensitivity of the double encoded experiment detected in a conventional way is still low because of the low filling factor of the fluid inside the NMR coil around the sample. We circumvent this problem by using the remote detection technique, which significantly increases the sensitivity, making it possible to do the measurements with gaseous fluids that have a low spin-density compared to liquids. We derive a model that enables us to extract a diffusion constant characterizing the diffusion rate through the membrane from the obtained correlation images. The double phase encoded MRI method is advantageous in any kind of diffusion studies, because the propagator of fluid molecules can directly be seen from the correlation image.     

Scaling of folding times with protein size

Current experimental data show a 9-orders-of-magnitude span in the folding times of proteins. Such a wide range is typically considered a direct consequence of the complexity in structural and sequence patterns of natural proteins. By using a database of 69 proteins and peptides analyzed experimentally, we observe that the folding time scales with the number of residues in the protein. The correlation coefficient is 0.74 or higher, and indicates that it is possible to predict the folding time of a protein with a precision of approximately 1.1 times decades from just its size. A simple thermodynamic analysis of this correlation suggests that the smallest proteins are expected to have very marginal free energy barriers to folding.     

Nanoelectrodes for intracellular measurements of reactive oxygen and nitrogen species in single living cells

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g. phagocytosis) and pathological conditions (e.g. cancer). The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this article, we survey recent advances in the localized measurements of ROS/RNS inside single cells and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.