Fast Exchange Rate Research Articles

Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.

Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.

Kinetic isotope effects for fast deuterium and proton exchange rates.

By monitoring the effect of deuterium decoupling on the decay of transverse (15)N magnetization in D-(15)N spin pairs during multiple-refocusing echo sequences, we have determined fast D-D exchange rates kD and compared them with fast H-H exchange rates kH in tryptophan to determine the kinetic isotope effect as a function of pH and temperature.

Automated Patch Clamp Meets High-Throughput Screening: 384 Cells Recorded in Parallel on a Planar Patch Clamp Module.

We have developed an automated patch clamp module for high-throughput ion channel screening, recording from 384 cells simultaneously. The module is incorporated into a laboratory pipetting robot and uses a 384-channel pipettor head for application of cells and compounds. The module contains 384 amplifier channels for fully parallel recordings using a digital amplifier. Success rates for completed experiments (1- to 4-point concentration-response curves for cells satisfying defined quality control parameters) of greater than 85% have been routinely achieved with, for example, HEK, CHO, and RBL cell lines expressing hNaV1.7, hERG, Kir2.1, GABA, or glutamate receptors. Pharmacology experiments are recorded and analyzed using specialized software, and the pharmacology of hNaV1.7 and hERG is described. Fast external solution exchange rates of <50 ms are demonstrated using Kir2.1. Short exposure times are achieved by stacking the external solutions inside the pipette of the robot to minimize exposure of the ligand on the receptor. This ensures that ligand-gated ion channels, for example, GABA and glutamate described in this report, can be reproducibly recorded. Stem cell-derived cardiomyocytes have also been used with success rates of 52% for cells that have a seal resistance of >200 MΩ, and recordings of voltage-gated Na+ and Ca2+ are shown.

Distribution of 223Ra and 224Ra in the Bo Sea embayment in Tianjin and its implication of submarine groundwater discharge

Submarine groundwater discharge (SGD) is now recognized as an important pathway between land and sea. In this study, in order to analyze the distribution of naturally occurring short-lived radium isotopes and the relative SGD effect in Bo Sea embayment, 223Ra and 224Ra were measured in three parts of the embayment with the radium-delayed coincidence counting (RaDeCC) system. Subsequently, the mixing process was studied by the calculation of diffusion coefficients (Kx and Kz) and advection velocities (Vx and Vz) based on the 2D advection-diffusion model. Additionally, the apparent residence ages and SGD flux were quantified based on the 224Ra and 223Ra activities. The results showed that the Ra activities exponentially decreased with the distance offshore, and both the Kx and Vx took the order of northern part > southern part > middle part. In vertical direction, there was the maximum value of Vz and minimum Kz in middle part and the maximum Kz and minimum Vz in southern part. The average ages for the northern, middle and southern parts were 4.28, 7.38 and 3.73 days, respectively. The final SGD flux yielded by 224Ra was 0.09, 0.01 and 0.03 m d−1 in the northern, middle and southern parts, respectively. The SGD flux yielded by 223Ra was 0.08, 0.01 and 0.03 m d−1 in northern, middle and southern parts, respectively. The result indicates that there is the fastest exchange rate and the biggest SGD flux in the southern part in Bo Sea embayment.

Annealing influence on the magnetostructural transition in Gd5Si1.3Ge2.7 thin films

Due to the emerging cooling possibilities at the micro and nanoscale, such as the fast heat exchange rate, the effort to synthesize and optimize the magnetocaloric materials at these scales is rapidly growing. Here, we report the effect of different thermal treatments on Gd5Si1.3Ge2.7 thin film in order to evaluate the correlation between the crystal structure, magnetic phase transition and magnetocaloric effect. For annealing temperatures higher than 773K, the samples showed a typical paramagnetic behavior. On the other hand, annealing below 773K promoted the suppression of the magnetostructural transition at 190K, while the magnetic transition around 249K is not affected. This magnetostructural transition extinction imparts reflected in the magnetocaloric behavior and resulted in a drastic decrease in the entropy change peak value. Nevertheless, an increase in 25% of the TC and an increasing ΔTFWHM from 23 to 49K of its operation temperature interval, ΔT, upon annealing, are crucial for future application in magnetic refrigeration.

CaSO4 Mineralization in Carboxy- and Amino-Functionalized Reverse Micelles Unravels Shape-Dependent Transformations and Long-Term Stabilization Pathways for Poorly Hydrated Nanophases (Bassanite)

Mineralization studies on calcium sulfates when compared to those on phosphates and carbonates are relatively rare despite the economic relevance of these sulfates. A series of recent findings, however, have renewed the interest in calcium sulfate mineralization. These studies, similar to those on calcium carbonates and phosphates, have shown a richer than previously thought scenario especially at the nanoscale. Here, we intentionally use confinement reactors with fast exchange rates (organically/inorganically functionalized reverse micelles) to rapidly establish pathways relevant to calcium sulfate mineralization in solution (including long-term stabilization). Furthermore, we rationalize all the results on the basis of an adsorption model with solubility inputs for inorganic additives. Thus, the typical bassanite nanofibers involving oriented attachment are observed either at weak-adsorption conditions or in the presence of less soluble inorganic dopants. At strong adsorption conditions, nanofibers are ...

Microdroplet fusion mass spectrometry for fast reaction kinetics

We investigated the fusion of high-speed liquid droplets as a way to record the kinetics of liquid-phase chemical reactions on the order of microseconds. Two streams of micrometer-size droplets collide with one another. The droplets that fused (13 μm in diameter) at the intersection of the two streams entered the heated capillary inlet of a mass spectrometer. The mass spectrum was recorded as a function of the distance x between the mass spectrometer inlet and the droplet fusion center. Fused droplet trajectories were imaged with a high-speed camera, revealing that the droplet fusion occurred approximately within a 500-μm radius from the droplet fusion center and both the size and the speed of the fused droplets remained relatively constant as they traveled from the droplet fusion center to the mass spectrometer inlet. Evidence is presented that the reaction effectively stops upon entering the heated inlet of the mass spectrometer. Thus, the reaction time was proportional to x and could be measured and manipulated by controlling the distance x. Kinetic studies were carried out in fused water droplets for acid-induced unfolding of cytochrome c and hydrogen-deuterium exchange in bradykinin. The kinetics of the former revealed the slowing of the unfolding rates at the early stage of the reaction within 50 μs. The hydrogen-deuterium exchange revealed the existence of two distinct populations with fast and slow exchange rates. These studies demonstrated the power of this technique to detect reaction intermediates in fused liquid droplets with microsecond temporal resolution.

A simple method for the synthesis of porous polymeric vesicles and their application as MR contrast agents.

Because of their low membrane permeability the use of polymeric vesicles in certain drug delivery and molecular imaging applications and as bioreactors is less than ideal. Here, we report a simple method to prepare porous polymeric vesicles that possess high membrane permeability. Specifically, porous vesicles were produced from the aqueous assembly of the diblock copolymer PEG-PBD, and the triblock copolymer PEG-PPO-PEG. It was found that PEG-PPO-PEG-doped polymersomes exhibited improved membrane permeability to molecules less than 5 kDa. Further, these porous vesicles retained molecules ≥10 kDa within their aqueous interiors with no significant leakage. To demonstrate its application, highly efficient magnetic resonance contrast agents were produced from porous polymersomes by encapsulating macromolecules labeled with gadolinium. Due to a fast water exchange rate with surrounding bulk water, these paramagnetic porous polymersomes exhibited higher r1 relaxivity compared with Gd-encapsulated vesicles with no pores. Due to their simplicity, the porous polymersomes prepared with this method are expected to have additional useful applications.

Double agents and secret agents: the emerging fields of exogenous chemical exchange saturation transfer and T2-exchange magnetic resonance imaging contrast agents for molecular imaging.

Two relatively new types of exogenous magnetic resonance imaging contrast agents may provide greater impact for molecular imaging by providing greater specificity for detecting molecular imaging biomarkers. Exogenous chemical exchange saturation transfer (CEST) agents rely on the selective saturation of the magnetization of a proton on an agent, followed by chemical exchange of a proton from the agent to water. The selective detection of a biomarker-responsive CEST signal and an unresponsive CEST signal, followed by the ratiometric comparison of these signals, can improve biomarker specificity. We refer to this improvement as a “double-agent” approach to molecular imaging. Exogenous T2-exchange agents also rely on chemical exchange of protons between the agent and water, especially with an intermediate rate that lies between the slow exchange rates of CEST agents and the fast exchange rates of traditional T1 and T2 agents. Because of this intermediate exchange rate, these agents have been relatively unknown and have acted as “secret agents” in the contrast agent research field. This review exposes these secret agents and describes the merits of double agents through examples of exogenous agents that detect enzyme activity, nucleic acids and gene expression, metabolites, ions, redox state, temperature, and pH. Future directions are also provided for improving both types of contrast agents for improved molecular imaging and clinical translation. Therefore, this review provides an overview of two new types of exogenous contrast agents that are becoming useful tools within the armamentarium of molecular imaging.

Lower Denticity Leading to Higher Stability: Structural and Solution Studies of Ln(III)–OBETA Complexes

The heptadentate ligand OBETA (2,2'-oxybis(ethylamine)-N,N,N',N'-tetraacetic acid) was reported to form complexes with Ln(3+) ions more stable than those formed by the octadentate and more popular congener EGTA (ethylene glycol O,O'-bis(ethylamine)-N,N,N',N'-tetraacetic acid). The structural features leading to this puzzling coordination paradox were investigated by X-ray diffraction, solution state NMR, molecular modeling, and relaxometric studies. The stability constant of Gd(OBETA) (log KGdL = 19.37, 0.1 M KCl) is 2 orders of magnitude higher than that of the higher denticity analogue Gd(EGTA) (log KGdL = 17.66, 0.1 M KCl). The half-lives (t1/2) for the dissociation reactions of Gd(OBETA) and Gd(EGTA) ([Cu(2+)]tot = 0.2 mM, [Cit(3-)]tot = 0.5 mM, [PO4(3-)]tot = 1.0 mM, and [CO3(2-)]tot = 25 mM at pH = 7.4 and 25 °C in 0.1 M KCl solution) are 6.8 and 0.63 h, respectively, reflecting the much higher inertness of Gd(OBETA) near physiological conditions. NMR studies and DFT calculations using the B3LYP functional and a large-core ECP indicate that the [Gd(OBETA)(H2O)2](-) complex most likely exists in solution as the Δ(λλ)(δδδδ)A/Λ(δδ)(λλλλ)A enantiomeric pair, with an activation free energy for the enantiomerization process of ∼40 kJ·mol(-1). The metal ion is nine-coordinate by seven donor atoms of the ligand and two inner-sphere water molecules. The X-ray crystal structure of [C(NH2)3]3[Lu(OBETA)(CO3)]·2H2O is in agreement with the predictions of DFT calculations, the two coordinated water molecules being replaced by a bidentate carbonate anion. The (1)H NMRD and (17)O NMR study revealed that the two inner-sphere water molecules in Gd(OBETA) are endowed with a relatively fast water exchange rate (kex(298) = 13 × 10(6) s(-1)). The higher thermodynamic stability and inertness of Ln(OBETA) complexes, peaking in the center of the 4f series, combined with the presence of two coordinated water molecules suggests that Gd(OBETA) is a promising paramagnetic probe for MRI applications.

Size and surface functionalization of iron oxide nanoparticles influence the composition and dynamic nature of their protein corona.

Nanoparticles (NPs) adsorb proteins when in the biological matrix, and the resulted protein corona could affect NP-cell interactions. The corona has a dynamic nature with the adsorbed proteins constantly exchanging with the free proteins in the matrix at various rates. The rapidly exchanging proteins compose the soft corona, which responds more dynamically to environment changes than the hard corona established by the ones with slow exchange rates. In the present study, the corona formed on the superparamagnetic iron oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation and ultracentrifugation, which rapidly differentiated the corona proteins based on their exchange rates. By varying the surface hydrophobicity of the SPIONs with a core size around 10 nm, we found out that, the more hydrophobic surface ligand attracted proteins with higher surface hydrophobicity and formed a more dynamic corona with a larger portion of the involved proteins with fast exchange rates. Increasing the core diameter of the SPIONs but keeping the surface ligand the same could also result in a more dynamic corona. A brief investigation of the effect on the cellular uptake of SPIONs using one selected corona protein, transferrin, was conducted. The result showed that, only the stably bound transferrin could significantly enhance cellular uptake, while transferrin bound in a dynamic nature had negligible impact. Our study has led to a better understanding of the relationship between the particle properties and the dynamic nature of the corona, which can help with design of nanomaterials with higher biocompatibility and higher efficacy in biosystems for biomedical applications.

Localization of MLH3 at the centrosomes.

Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLβ, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.

The oxygen sensitive properties of (LaBa)Co2O5+δ thin films fabricated by polymer-assisted deposition technique

(LaBa)Co2O5+δ (LBCO) thin films were successfully fabricated on Si (001) substrates by polymer-assisted deposition method. Microstructures were examined by X-ray diffraction technique, which confirmed the films were a single phase, pseudo cubic structure. The electrical transport properties of the films were investigated by the temperature dependence of films resistance, which suggested that LBCO films have typical semiconductor properties. After circle tests, the oxygen–hydrogen response rate did not show obvious variation in the specific temperature environment of 580 °C. This demonstrates that the LBCO thin films have a superior stability in both oxygen and hydrogen (6 % H2, 94 % N2) environment. Simultaneously, the drastic changes of films resistance (from ~106 to ~102 Ω) with the switch of redox (O2–H2) environment within such a short time (~2.2 s) indicated that LBCO films have an excellent oxygen sensitive property and extraordinary fast surface exchange rate.

Molecular Recognition of Epothilones by Microtubules and Tubulin Dimers Revealed by Biochemical and NMR Approaches

The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

Determination of Electrode Oxygen Transport Kinetics Using Electrochemical Impedance Spectroscopy Combined with Three-Dimensional Microstructure Measurement: Application to Nd2NiO4+δ

Oxygen reduction kinetic parameters – oxygen ion diffusion Dδ, molar surface exchange rate ℜO and surface exchange coefficient k – were determined for porous Nd2NiO4+δ solid oxide fuel cell cathodes as a function of temperature and oxygen partial pressure by analyzing electrochemical impedance spectroscopy data using the Adler-Lane-Steele model. Electrode microstructural data used in the model calculations were obtained by three-dimensional focused ion beam-scanning electron microscope tomography. Cathodes were fabricated using Nd2NiO4+δ powder derived from a sol-gel method and were tested as symmetrical cells with LSGM electrolytes. The oxygen surface exchange rate exhibited a power-law dependency with oxygen partial pressure, whereas the oxygen diffusivity values obtained varied only slightly. The present analysis suggests that the O-interstitial diffusion has a bulk transport path, whereas the surface exchange process involves dissociative adsorption on surface sites followed by O-incorporation. For Nd2NiO4+δ at 700°C and 0.2 atm oxygen pressure, Dδ = 5.6·10−8 cm2s−1, ℜO = 2.5·10−8 mol·cm−2 s−1. The present Dδ and ℜO values and their activation energies are slightly different to those previously reported for Nd2NiO4+δ using other measurement methodologies, and lower than typical state-of-the-art Co-rich perovskites. However, the average kδ = 1.0 10−5 cm·s−1 at 700°C is comparable to those of fast oxygen exchange rate perovskites.

Fluid distribution and pore wettability of monolithic carbon xerogels measured by 1H NMR relaxation

The pore wettability of two types of carbon xerogels exhibiting micro- and mesopores with different sizes was investigated using 1H NMR relaxation. These nanomaterials are great candidates as active material for gas diffusion electrodes in mobile energy storage devices. The distribution of fluids in these electrodes is a critical parameter determining the electrode performance. Using 1H NMR relaxation information about the wettability of micro- as well as mesopores is determined simultaneously. Two important solvents for electrolytes, water and dimethyl sulfoxide (DMSO), were studied for different filling states inside the porous structure. While for water no discrimination between micro- and mesopores could be achieved because of its fast exchange rate between the pores, the measurements with DMSO allow for discriminating between solvent in micro- and mesopores. Thus, the wettability of DMSO in both pore regimes was determined by calculating the surface relaxation strength. It turned out that the wettability of the porous structure is dependent on the differences in the pore sizes between micro- and mesopores. At low filling states both pore regimes compete for the remaining fluid leading to a nonuniform distribution. The pores with the better wettability keep the complete surface in contact with the bulk fluid.

The Hanes‐Woolf linear QUESP method improves the measurements of fast chemical exchange rates with CEST MRI

Contrast agents for chemical exchange saturation transfer MRI often require an accurate measurement of the chemical exchange rate. Many analysis methods have been reported that measure chemical exchange rates. Additional analysis methods were derived as part of this study. This report investigated the accuracy and precision of each analysis method. Chemical exchange saturation transfer spectra were simulated using the Bloch-McConnell equations modified for chemical exchange. Chemical exchange saturation transfer spectra of iopromide were obtained with a range of saturation times, saturation powers, and concentrations. These simulated and experimental results were used to estimate the chemical exchange rate using the QUESP, QUEST, Omega Plot (LB-QUESP), EH-QUESP, HW-QUESP, LB-Conc, EH-Conc, and HW-Conc methods. Bloch fitting produced the most precise estimates of chemical exchange rates, although substantial expertise and computation time were required to achieve these results. Of the more simplistic analysis methods, the HW-QUESP method produced the most accurate and precise estimates of fast exchange rates. The QUEST and LB-QUESP methods produced the most accurate estimates of slow exchange rates, especially with samples that have short T(1w) relaxation times. HW-QUESP is a simplistic analysis method that should be used when fast chemical exchange rates need to be estimated from chemical exchange saturation transfer MRI results.

Exchange rate pass-through: evidence from the Syrian economy

This paper studies the exchange rate pass-through in the Syrian economy over the period 1990Q1-2009Q4. To this end it constructs a cost of imports indicator which is used in an Autoregressive Distributed Lag (ARDL) approach. The findings point to a high and fast exchange rate pass-through effect. As a consequence, Syrian macro-economic performance is very sensitive to international price evolutions as well as depreciations of the Syrian Pound.

Characterization of mitotane (o,p′-DDD) – cyclodextrin inclusion complexes: Phase-solubility method and NMR

Mitotane (o,p'-dichlorodimethyl dichloroethane [o,p'-DDD]) is used for the treatment of adrenocortical cancer and occasionally Cushing's syndrome. This drug is very poorly soluble in water, and following oral administration, approximately 60% of the dose is recovered in the feces unaltered. The preparation of a soluble formulation (i.e. by complexation with cyclodextrins) with improved bioavailability is the aim of this work. The inclusion of mitotane in methyl-ß-cyclodextrins was studied using both phase-solubility methods and NMR experiments. To elucidate the inclusion mechanism, o,p'-DDD was compared to its regioisomer (i.e. p,p'-DDD). It was demonstrated that two dimethyl-ß-cyclodextrins (DMßCD) can complex with the aromatic rings. From the phase-solubility diagrams, we observe that both cases are very different: K(1:1) is between 37 000 and 85 000 mol.l(-1), whereas K(1:2) is between 5.3 and 32 mol.l(-1). The NMR experiments confirmed the inclusion but it also gave an insight into the kinetics of the dissociation: the ortho-chloro moiety is in slow exchange on the NMR time scale, whereas the para-chloro moiety is in fast exchange rate.

1H and 17O NMR Relaxometric and Computational Study on Macrocyclic Mn(II) Complexes

Herein we report a detailed 1H and 17O relaxometric investigation of Mn(II) complexes with cyclen-based ligands such as 2-(1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (DO1A), 2,2'-(1,4,7,10-tetraazacyclododecane-1,4-diyl)diacetic acid (1,4-DO2A), 2,2'-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (1,7-DO2A), and 2,2',2"-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DO3A). The Mn(II) complex with the heptadentate ligand DO3A does not have inner sphere water molecules (q = 0), and therefore, the metal ion is most likely seven-coordinate. The hexadentate DO2A ligand has two isomeric forms: 1,7-DO2A and 1,4-DO2A. The Mn(II) complex with 1,7-DO2A is predominantly six-coordinate (q = 0). In aqueous solutions of [Mn(1,4-DO2A)], a species with one coordinated water molecule (q = 1) prevails largely, whereas a q = 0 form represents only about 10% of the overall population. The Mn(II) complex of the pentadentate ligand DO1A also contains a coordinated water molecule. DFT calculations (B3LYP model) are used to obtain information about the structure of this family of closely related complexes in solution, as well as to determine theoretically the 17O and 1H hyperfine coupling constants responsible for the scalar contribution to 17O and 1H NMR relaxation rates and 17O NMR chemical shifts. These calculations provide 17O A/ħ values of ca. 40 × 10(6) rad s(-1), in good agreement with experimental data. The [Mn(1,4-DO2A)(H2O)] complex is endowed with a relatively fast water exchange rate (k(ex)298 = 11.3 × 10(8) s(-1)) in comparison to the [Mn(EDTA)(H2O)]2- analogue (k(ex)298 = 4.7 × 10(8) s(-1)), but about 5 times lower than that of the [Mn(DO1A)(H2O)]+ complex (k(ex)298 = 60 × 10(8) s(-1)). The water exchange rate measured for the latter complex represents the highest water exchange rate ever measured for a Mn(II) complex.