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Regulator of G protein signaling 4 (RGS4) is an intracellular protein that binds to the Gα subunit ofheterotrimeric G proteins and aids in terminating G protein coupled receptor signaling. RGS4 has been implicated in pain, schizophrenia, and the control of cardiac contractility. Inhibitors of RGS4 have been developed but bind covalently to cysteine residues on the protein. Therefore, we sought to identify alternative druggable sites on RGS4 using mixed-solvent molecular dynamics simulations, which employ low concentrations of organic probes to identify druggable hotspots on the protein. Pseudo-ligands were placed in consensus hotspots, and perturbation with normal mode analysis led to the identification and characterization of a putative allosteric site, which would be invaluable for structure-based drug design of non-covalent, small molecule inhibitors. Future studies on the mechanism of this allostery will aid in the development of novel therapeutics targeting RGS4.

The position-dependent diffusion coefficient along with free energy profile are important parameters needed to study mass transport in heterogeneous systems such as biological and polymer membranes, and molecular dynamics (MD) calculation is a popular tool to obtain them. Among many methodologies, the Marrink-Berendsen (MB) method is often employed to calculate the position-dependent diffusion coefficient, in which the autocorrelation function of the force on a fixed molecule is related to the friction on the molecule. However, the diffusion coefficient is shown to be affected by the period of the removal of the center-of-mass velocity, which is necessary when performing MD calculations using the Ewald method for Coulombic interaction. We have clarified theoretically in this study how this operation affects the diffusion coefficient calculated by the MB method, and the theoretical predictions are proven by MD calculations. Therefore, we succeeded in providing guidance on how to select an appropriate the period of the removal of the center-of-mass velocity in estimating the position-dependent diffusion coefficient by the MB method. This guideline is applicable also to the Woolf-Roux method.

This study evaluated an electronic-nose (e-nose) sensor in combination with support vector machine (SVM) modeling for predicting the decomposition state of four types of fish fillets: mahi-mahi, croaker, red snapper, and weakfish. The National Seafood Sensory Expert scored fillets were thawed, 10-g portions were weighed into glass jars which were then sealed, and the jars were held at approximately 30°C to allow volatile components to be trapped and available for analysis. The measurement of the sample vial headspace was performed with an e-nose device consisting of nanocomposite, metal oxide semiconductor (MOS), electrochemical, and photoionization sensors. Classification models were then trained based on the sensory grade of each fillet, and the e-nose companion chemometric software identified that eight MOS were the most informative for determining a sensory pass from sensory fail sample. For SVM, the cross-validation (CV) correct classification rates for mahi-mahi, croaker, red snapper, and weakfish were 100%, 100%, 97%, and 97%, respectively. When the SVM prediction performances of the eight MOS were evaluated using a calibration-independent test set of samples, correct classification rates of 93-100% were observed. Based on these results, the e-nose measurements coupled with SVM models were found to be potentially promising for predicting the spoilage of these four fish species. PRACTICAL APPLICATION: This report describes the application of an electronic-nose sensor as a potential rapid and low-cost screening method for fish spoilage. It could provide regulators and stakeholders with a practical tool to rapidly and accurately assess fish decomposition.

Hydrogen is one of the most promising sustainable energy carriers for its high gravimetric energy density and abundance. Nowadays, hydrogen production and storage are the main constraints for its commercialization. As a current research focus, hydrogen production from methanol-water reforming, especially at low temperature, is particularly important. In this study, a novel reaction path for low-temperature methanol reforming through synergistic catalysis was developed. Alcohol dehydrogenase (ADH) and coenzyme I (nicotinamide adenine dinucleotide, NAD+ ) were employed for methanol catalytic dehydrogenation at low temperature, which could generate formaldehyde and reductive coenzyme I (NADH). Covalent triazine framework-immobilized ruthenium complex (Ru-CTF) was prepared afterwards. On one hand, the catalyst exhibited high activity for the formaldehyde-water shift reaction to generate hydrogen and carbon dioxide. On the other hand, the NADH dehydrogenation was also catalyzed by the Ru-CTF, producing NAD+ and hydrogen. Additionally, the catalyst also showed high biocompatibility with ADH. Through the synergistic effect of the above two main processes, methanol could be converted into hydrogen and carbon dioxide stably at low temperature for more than 96 h. The hydrogen production rate was dependent on the pH of the reaction solution as well as the ADH dosage. A hydrogen production rate of 157 mmol h-1 mol-1 Ru was achieved at the optimum pH (8.1). Additionally, the hydrogen production rate increased linearly with the ADH dosage, reaching 578 mmol h-1 mol-1 Ru when the ADH dosage was 180 U at 35 °C. This research could not only help overcome the difficulties for methanol reforming near room temperature but also give new inspiration for designing new reaction pathways for methanol reforming.

Amyloid fibrils are ordered aggregates that may be formed from disordered, partially unfolded, and fragments of proteins and peptides. There are several diseases, which are due to the formation and deposition of insoluble β‐sheet protein aggregates in various tissue, collectively known as amyloidosis. Here, we have used bovine α‐lactalbumin as a model protein to understand the mechanism of amyloid fibril formation at pH 1.6 and 65°C under non‐reducing conditions. Amyloid fibril formation is confirmed by Thioflavin T fluorescence and atomic force microscopy (AFM). Our finding demonstrates that hydrolysis of peptide bonds occurs under these conditions, which results in nicking and fragmentation. The nicking and fragmentation have been confirmed on non‐reducing and reducing gel. We have identified the fragments by matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry. The fragmentation may initiate nucleation as it coincides with AFM images. Conformational changes associated with monomer resulting in fibrillation are shown by circular dichroism and Raman spectroscopy. The current study highlights the importance of nicking and fragmentation in amyloid fibril formation, which may help understand the role of acidic pH and proteolysis under in vivo conditions in the initiation of amyloid fibril formation.

Water oxidation is a bottleneck in artificial photosynthesis that impedes its practicality for solar energy conversion and utilization. It is highly desired to significantly improve the efficacy of the existing catalysts or to rationally design new catalysts with improved performance. We report a novel conjugated and sulfone containing polyimide as a metal-free photocatalyst synthesized via a two-step method: (i) synthesis of precursor poly(amic acid) (PAA) (ii) solvothermal synthesis of polyimide through thermal imidization. The synthesis of the polyimide photocatalyst was demonstrated by the amide linkage in the FTIR spectrum. The obtained photocatalyst was semicrystalline in nature and possessed sheet-like morphology as illustrated by the diffraction pattern and the electron micrographic images, respectively. The thermogravimetric analysis of the polyimide nanosheets validated a thermally stable structure. The DFT calculations were performed which showed a suitable HOMO band position, favorable for water oxidation. The photoelectrocatalytic (PEC) performance of the polyimide nanosheets evaluated by studying water oxidation reaction without any sacrificial agent under 1-SUN showed enhanced PEC performance and good stability towards water oxidation at 0 V versus SCE.

Multiple impeller reactors are widely used due to their advanced gas utilization and an increased volumetric mass transfer coefficient. However, with the application of Rushton impellers, gas dispersion efficiency varies between the bottom and the upper impeller levels. The present study analyzes the individual flow regime, power input, and gas hold-up in each compartment of a reactor equipped with four Rushton impellers. The results indicate that the pre-dispersion of the air introduced by the bottom impeller (up to 80%) plays a key role in a better gas retention efficiency of the upper impellers (>300%) and leads to a shift of the cavity and flooding lines in the flow map (Fr- vs Fl-Number) of the upper impellers. A novel analysis of the bubble flow in the dispersed state via a two-phase LES-based CFD model reveals that a more homogenous distribution of air bubbles in the upper compartments leads to high compartment gas hold-up values, but fewer bubbles in the vicinity of the impellers. The measured and simulated data of this study indicate that the upper impellers' efficiency mostly depends on the flow regime of and the pre-dispersion by the bottom impeller rather than on the upper impellers' flow regimes. These results contribute to the understanding of essential mixing processes and scaling of aerated bioreactors.

The liquid-liquid phase separation (LLPS) of Tau has been postulated to play a role in modulating the aggregation property of Tau, a process known to be critically associated with the pathology of a broad range of neurodegenerative diseases including Alzheimer's Disease. Tau can undergo LLPS by homotypic interaction through self-coacervation (SC) or by heterotypic association through complex-coacervation (CC) between Tau and binding partners such as RNA. What is unclear is in what way the formation mechanisms for self and complex coacervation of Tau are similar or different, and the addition of a binding partner to Tau alters the properties of LLPS and Tau. A combination of in vitro experimental and computational study reveals that the primary driving force for both Tau CC and SC is electrostatic interactions between Tau-RNA or Tau-Tau macromolecules. The liquid condensates formed by the complex coacervation of Tau and RNA have distinctly higher micro-viscosity and greater thermal stability than that formed by the SC of Tau. Our study shows that subtle changes in solution conditions, including molecular crowding and the presence of binding partners, can lead to the formation of different types of Tau condensates with distinct micro-viscosity that can coexist as persistent and immiscible entities in solution. We speculate that the formation, rheological properties and stability of Tau droplets can be readily tuned by cellular factors, and that liquid condensation of Tau can alter the conformational equilibrium of Tau.

International audience; These last decades, it has been widely assumed that 18-crown-6-ether (CE) plays a spectator role during the chemical processes occurring in isolated host-guest complexes between peptides or proteins and CE, after activation in mass spectrometers. Our present experimental and theoretical results challenge this hypothesis by showing that CE can abstract a proton or a protonated molecule from protonated peptides after activation by collisions in argon or electron capture/transfer. Furthermore, thanks to comparison between experimental and calculated values of collision cross-sections, we demonstrate that CE can change binding site after electron transfer. We also propose detailed mechanisms for these processes.

During fill-finish manufacturing, therapeutic proteins may aggregate or form subvisible particles in response to the physical stresses encountered within filling pumps. Understanding and quantitating this risk is important since filling may be the last unit operation before the patient receives their dose. We studied particle formation from lab-scale to manufacturing-scale using sensitive and robust protein formulations. Filling experiments with a ceramic rotary piston pump were integrated with a rinse-stripping method to investigate the relationship between protein adsorption and particle formation. For a sensitive protein, multilayer film formation on the piston surface correlated with high levels of subvisible particles in solution. For a robust protein formulation, adsorption and subvisible particle formation were minimal. These results support an aggregation mechanism that is initiated by adsorption to pump surfaces and propagated by mechanical and/or hydrodynamic disruption of the film. The elemental analysis confirmed that ceramic wear debris remained at trace levels and did not contribute appreciably to protein aggregation.

Statistical methods were used to provide insight into a polymer complex system composed of lysozyme and alginate to quantify the effects of such parameters as pH, and ionic composition of the mixing solution on the properties of the complexes including composition, particle diameter, and zeta potential. Various crosslinkers (calcium, barium, iron[III], and bovine serum albumin), were used with lysozyme for complex formation to investigate the effect of crosslinker charge density on protein release kinetics, modelled using ktn . Multivariate statistical analysis showed that the kinetic parameters associated with the release were, not surprisingly highly dependent on the ionic strength of the release media, with higher ionic strength leading to faster release. The release parameter k was also shown to depend on the protein properties (size, ionic strength) while n was slightly, but not statistically dependent on the charge density of the crosslinker demonstrating that the nature of the crosslinker had minimal impact on drug release. The multivariate statistical has the potential to be used for optimization of the complexes and prediction of physical properties and degradation rates.

In this study, five adsorption isotherm models, that is, the Langmuir, Freundlich, Langmuir-Freundlich, Temkin and Brunauer-Emmett-Teller isotherms, were utilized for the analysis of the experimental adsorption data for six classes of poly(N-isopropylacrylamide)-based thermo-responsive copolymer-grafted Sepharose Fast Flow sorbents of different copolymer compositions with two structurally related proteins, namely bovine holo-lactoferrin and bovine holo-transferrin at 20 and 50°C. The experimental data for bovine holo-lactoferrin could be mathematically fitted to the Freundlich and Temkin isotherms when the protein feed concentrations were in the range of 1-40 mg/mL at both 20 and 50°C. Similar analysis of the binding of the homologous protein, bovine holo-transferrin, to the same thermo-responsive copolymer-grafted sorbents revealed that the experimental data could be fitted to the Langmuir, Freundlich and Temkin isotherms with coefficients of determination value over 0.90.

Hydrogen sulfide (H2 S) is recognized as an endogenous gaseous signaling agent in many biological activities. Lysosomes are the main metabolic site and play a pivotal role in cells. Herein, we designed and synthesized two new fluorescent probes BDP-DNBS and BDP-DNP with a BODIPY core to distinguish H2 S. The sensing mechanism is based on the inhibition-recovery of the photo-induced electron transfer (PET) process. Through comparing the responsive behaviors of the two probes toward H2 S, BDP-DNBS showed a fast response time (60 s), low limit of detection (LOD, 51 nM), high sensitivity and selectivity. Moreover, the reaction mechanism was demonstrated by mass spectrometry and fluorescence off-on mechanism was proved by density functional theory (DFT). Significantly, confocal fluorescence imaging indicated that BDP-DNBS was successfully used to visualize H2 S in lysosomes in living HeLa cells.

We propose a micromixer for obtaining better efficiency of vortex induced electroosmotic mixing of non-Newtonian bio-fluids at a relatively higher flow rate, which finds relevance in many biomedical and biological applications. To represent the rheology of non-Newtonian fluid, we consider the Carreau model in this study, while the applied electric field drives the constituent components in the micromixer. We show that the spatial variation of the applied field, triggered by the topological change of the bounding surfaces, upon interacting with the non-uniform surface potential gives rise to efficient mixing as realized by the formation of vortices in the proposed micromixer. Also, we show that the phase-lag between surface potential leads to the formation of asymmetric vortices. This behavior offers better mixing performance following the appearance of undulation on the flow pattern. Finally, we establish that the assumption of a point charge in the paradigm of electroosmotic mixing, which is not realistic as well, under-predicts the mixing efficiency at higher amplitude of the non-uniform zeta potential. The inferences of the present analysis may guide as a design tool for micromixer where rheological properties of the fluid and flow actuation parameters can be simultaneously tuned to obtain phenomenal enhancement in mixing efficiency.

Integrated continuous downstream processes with process analytical technology offer a promising opportunity to reduce production costs and increase process flexibility and adaptability. In this case study, an integrated continuous process was used to purify a recombinant protein on laboratory scale in a two-system setup that can be used as a general downstream setup offering multi-product and multi-purpose manufacturing capabilities. The process consisted of continuous solvent/detergent virus inactivation followed by periodic countercurrent chromatography in the capture step, and a final chromatographic polishing step. A real-time controller was implemented to ensure stable operation by adapting the downstream process to external changes. A concentration disturbance was introduced to test the controller. After the disturbance was applied, the product output recovered within 6 hours, showing the effectiveness of the controller. In a comparison of the process with and without the controller, the product output per cycle increased by 27%, the resin utilization increased from 71.4% to 87.9%, and the specific buffer consumption was decreased by 21% with the controller, while maintaining a similar yield and purity as in the process without the disturbance. In addition, the integrated continuous process outperformed the batch process, increasing the productivity by 95% and the yield by 28%. (Less)

This communication reports the beneficial effects of co-gasification of biomass and residual oil to produce syngas. In this regard, various blends of glucose (a biomass surrogate) to vacuum gas oil (VGO) have been employed to investigate the synergic effects on the gasification process. The non-isothermal co-gasification experiments were conducted in a thermogravimetric analyzer at different heating rates and gasifying agents. The analysis showed that the co-gasification rate increased with the increase of glucose content in the feedstock. The presence of the oxygen in the biomass molecules helped the overall gasification process. The maximum gasification rate of 42.70 wt/min (DTGmax ) was observed with 25 wt% glucose containing sample. The use of gasifying agents appeared to have some influence, especially during high temperature gasification of the glucose-VGO blends. At a same gasification temperature, the co-gasification rate of glucose-VGO blends were found to be 125.7 wt/min and 98.59 wt%/min for N2 and CO2 , respectively. The kinetics of the co-gasification of glucose-VGO blends was conducted based on modified random pore model using TGA experimental data and implemented in MATLAB. The estimated activation energy and rate constants were found to be consistent to the observed co-gasification rates. The apparent activation energies of co-gasification of VGO/biomass blends with different weight percentages shows values ranging 60.56-48.25 kJ/mol. The kinetics analysis suggested that the addition of biomass helped to increase the reaction rate by lowering the activation energy required for accomplishing the reactions compared with petroleum carbonaceous feedstocks. The reaction rate constants isotherms are plotted to show that the k-values are exhibiting similar trends at moderate heating rates between 20 and 60 °C/min. This remark arises due to the nature of the reactions involved which are considered to be inherently similar in this range of heating rate.

A new method was proposed for increasing the capture chromatography process efficiency, linear flow‐velocity gradient (LFG). The method uses a linear decreasing flow‐velocity gradient with time during the sample loading. The initial flow velocity, the final flow velocity and the gradient time are the parameters to be tuned. We have developed a method for determining these parameters by using the total column capacity and the total loaded amount as a function of time. The capacity can be calculated by using the relationships between dynamic binding capacity (DBC) and residence time. By leveraging the capacity, loading amount, and the required conditions, the optimum LFG can be designed. The method was verified by ion‐exchange and protein A chromatography of monoclonal antibodies (mAbs). A two‐fold increase in the productivity during the sample loading was possible by LFG compared with the constant flow‐velocity (CF) operation. LFG was also applied to a 4‐column continuous process. The simulation showed that the cost of resin per unit amount of processed mAbs can be reduced by 13% while 1.4 times enhancement in productivity was preserved after optimization by LFG compared to CF. The process efficiency improvement is more pronounced when the isotherm is highly favorable and the loading volume is large., Linear flow‐velocity gradient (LFG) is developed to increase the efficiency of the capture chromatography process for proteins (mAbs). By applying a decreasing LFG to the loading step, dynamic binding capacity increases with time. The optimal LFG can be determined by achieving the maximum capacity utilization. Although LFG is effective for the batch operation, it can be applied to the continuous process. A 1.4 fold increase in productivity and a 13% reduction in resin cost are shown by comparing LFG with the constant flow‐velocity operation in a 4‐column continuous chromatography process of mAbs.

Globular proteins exhibit dielectrophoresis (DEP) responses in experiments where the applied field gradient factor ∇E2 appears far too small, according to standard DEP theory, to overcome dispersive forces associated with the thermal energy kT of disorder. To address this a DEP force equation is proposed that replaces a previous empirical relationship between the macroscopic and microscopic forms of the Clausius-Mossotti factor. This equation relates the DEP response of a protein directly to the dielectric increment de+ and decrement de− that characterize its v-dispersion at radio frequencies, and also indirectly to its intrinsic dipole moment by way of providing a measure of the protein's effective volume. A parameter Gpw, taken as a measure of cross-correlated dipole interactions between the protein and its water molecules of hydration, is included in this equation. For 9 of the 12 proteins, for which an evaluation can presently be made, Gpw has a value of ≈4600 ± 120. These conclusions follow an analysis of the failure of macroscopic dielectric mixture (effective medium) theories to predict the dielectric properties of solvated proteins. The implication of a polarizability greatly exceeding the intrinsic value for a protein might reflect the formation of relaxor ferroelectric nanodomains in its hydration shell.

Xylitol is commonly known as one of the top platform intermediates for biomass conversion. Catalytic deoxygenation of xylitol provides an atomic and energetic efficient way to produce a variety of renewable chemicals including ethylene glycol, 1,2-propanediol, lactic acid and 1,4-anhydroxylitol. Despite a few initial attempts in converting xylitol into those products, improving catalyst selectivity towards C-O and C-C cleavage reactions remains a grand challenge in this area. To our best knowledge, there is lack of comprehensive review to summarize the most recent advances on catalyst design and mechanisms in deoxygenation of xylitol, offering important perspective into future development of xylitol transformation technologies. Therefore, in this mini-review, we have critically discussed the conversion routes involved in xylitol deoxygenation over solid catalyst materials, the nanostructures of supported metal catalysts for C-H, C-C and C-O bond cleavage reactions, and mechanistic investigation for xylitol conversion. The outcome of this work provides new insights into rational design of effective deoxygenation catalyst materials for upgrading of xylitol and future process development in converting hemicellulosic biomass.

FFLUX is a biomolecular force field under construction, based on Quantum Chemical Topology (QCT) and machine learning (kriging), with a minimalistic and physically motivated design. A detailed analysis of the forces within the kriging models as treated in FFLUX is presented, taking as a test example a liquid water model. The energies of topological atoms are modelled as 3Natoms-6 dimensional potential energy surfaces, using atomic local frames to represent the internal degrees of freedom. As a result, the forces within the kriging models in FFLUX are inherently N-body in nature where N refers to Natoms. This provides a fuller picture that is closer to a true quantum mechanical representation of interactions between atoms. The presented computational example quantitatively showcases the non-negligible (as much as 9 %) three-body nature of bonded forces and angular forces in a water molecule. We discuss the practical impact on the pressure calculation with N-body forces and periodic boundary conditions (PBC) in molecular dynamics, as opposed to classical force fields with two-body forces. The equivalence between the PBC-related correction terms in the general virial equation is shown mathematically.

Determining the fate of nitroaromatic compounds (NACs) in the environment requires the use of predictive models for compounds and conditions for which experimental data are insufficient. Previous studies have developed linear free energy relationships (LFERs) that relate the thermodynamic energy of NAC reduction to its corresponding rate constant. Here we present a comprehensive LFER that incorporates both the reduction and oxidation half reactions through Quantum Chemically (QC) calculated energies. This article is protected by copyright. All rights reserved.

Protein–protein interactions (PPIs) in all the molecular aspects that take place both inside and outside cells. However, determining experimentally the structure and affinity of PPIs is expensive and time consuming. Therefore, the development of computational tools, as a complement to experimental methods, is fundamental. Here, we present a computational suite: MODPIN, to model and predict the changes of binding affinity of PPIs. In this approach we use homology modeling to derive the structures of PPIs and score them using state‐of‐the‐art scoring functions. We explore the conformational space of PPIs by generating not a single structural model but a collection of structural models with different conformations based on several templates. We apply the approach to predict the changes in free energy upon mutations and splicing variants of large datasets of PPIs to statistically quantify the quality and accuracy of the predictions. As an example, we use MODPIN to study the effect of mutations in the interaction between colicin endonuclease 9 and colicin endonuclease 2 immune protein from Escherichia coli. Finally, we have compared our results with other state‐of‐art methods.

The objective of this study is to fabricate customized dosage forms using extrusion-based 3D printing for the sustained delivery of theophylline. The therapeutic paste was prepared by combining various doses of theophylline (0, 75, 100, and 125 mg) with different concentrations of methylcellulose (MC) A4M (8, 10, and 12%). The paste was then 3D printed into semisolid tablets under optimized printing conditions. The rheological properties of printing pastes were related to the 3D printability. Our results indicated that to be 3D printed using the current platform, the storage modulus (G') of the printing paste should be higher than the loss modulus (G″) during the frequency sweep (0.1-600 rad/s), and the tan δ should fall in the range of 0.25-0.27 at 0.63 rad/s. The printed tablets formulated with 10% MC showed the highest overall quality, considering the aspects of resolution, texture, and shape retention regardless of the dosage. The scanning electron microscopy images indicated that the cross-linked structure of MC A4M formed the microscale porous microstructure, which has the potential to embed the theophylline, thus delayed the release through the barrier effect. The in vitro dissolution test revealed that the 3D printed tablets exhibited a sustained release during the first 12 hr. The findings in this study will support the development of customized, personalized medicine with improved efficacy.

Using silk fibroin as the base material, the drug-loaded microspheres are prepared by an emulsification method. In order to determine the drug-loading and drug-release performance parameters of the microspheres, the central composite design method is used to design and investigate the effects of the parameters of the microsphere preparation process, such as the oil-water ratio, stirring temperature, and stirring rate, on the microsphere particle size, drug-loading rate, and drug release rate. The "overall desirability" is taken as a comprehensive evaluation index, and the response surface method (RSM) and genetic algorithm-backpropagation (GA-BP) neural network GA-BP model are used to predict and evaluate the parameters of the drug-loaded microsphere preparation process. The root-mean-square error values obtained from the RSM and BP-GA model experiments are 0.000325 and 0.00022, respectively. The results show that the BP-GA model has better prediction accuracy and optimization ability than the RSM. The optimal microsphere preparation process conditions were determined to be as follows: a water-oil ratio of 10:1, at a temperature of 45°C with stirring at a speed of 400 rpm, the particle size of the microspheres is 1.392 μm, the drug-loading rate is 3.218%, and the drug release rate is 51.991%. The results of this study indicate that this approach is an effective method for the optimization of the parameters of the drug-loaded microsphere preparation process.

The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.

Cellular signaling is regulated by the assembly of proteins into higher‐order complexes. Bottom‐up creation of synthetic protein assemblies, especially asymmetric complexes, is highly challenging. Presented here is the design and implementation of asymmetric assembly of a ternary protein complex facilitated by Rosetta modeling and thermodynamic analysis. The wild‐type symmetric CT32–CT32 interface of the 14‐3‐3–CT32 complex was targeted, ultimately favoring asymmetric assembly on the 14‐3‐3 scaffold. Biochemical studies, supported by mass‐balance models, allowed characterization of the parameters driving asymmetric assembly. Importantly, our work reveals that both the individual binding affinities and cooperativity between the assembling components are crucial when designing higher‐order protein complexes. Enzyme complementation on the 14‐3‐3 scaffold highlighted that interface engineering of a symmetric ternary complex generates asymmetric protein complexes with new functions., Made to order: A designed asymmetric assembly creates novel synthetic higher‐order protein assemblies. A symmetric ternary protein complex is formed through stabilizer (red) mediated peptide (green) binding to a scaffold (grey). Modulation of the interface between the peptides (yellow, blue) leads to asymmetric protein self‐sorting. This elucidation of higher‐order protein structures provides insights into protein assembly and the connected role of scaffold proteins.

The Hill equation, which models the cooperative ligand‐receptor binding equilibrium, turns out to be useful in modeling the progression of infectious disease outbreaks such as CoViD‐19. The equation fits well the data for total and daily case numbers, allows tentative predictions for the half‐point and end point of the epidemic, and presents a mathematical characterization of how social interventions “flatten the curve” of the disease progression.

We investigated the effect of the cation-π interaction on the susceptibility of a tryptophan model system toward interaction with singlet oxygen, that is, type II photooxidation. The model system consists of two indole units linked to a lariat crown ether to measure the total rate of removal of singlet oxygen by the indole units in the presence of sodium cations (i.e. indole units subject to a cation-π interaction) and in the absence of this interaction. We found that the cation-π interaction significantly decreases the total rate of removal of singlet oxygen (kT ) for the model system, that is, (kT = 2.4 ± 0.2) × 108 m-1 s-1 without sodium cation vs (kT = 6.9 ± 0.9) × 107 m-1 s-1 upon complexation of sodium cation to the crown ether. Furthermore, we found that the indole moieties undergo type I photooxidation processes with triplet excited methylene blue; this effect is also inhibited by the cation-π interaction. The chemical rate of reaction of the indole groups with singlet oxygen is also slower upon complexation of sodium cation in our model system, although we were unable to obtain an exact ratio due to differences of the chemical reaction rates of the two indole moieties.

Mechanically assisted crevice corrosion may disrupt passive oxide films on medical alloys and lead to rapid repassivation reactions which generate corrosion currents and shifts in electrode potential due to the non-equilibrium nature of the reactions and the transient unbalancing of anodic and cathodic reactions. This study presents a theoretical approach to predict currents and voltages over time utilizing the concepts of heredity integrals, area-dependent surface impedance, contact mechanics and the high field physics of oxide repassivation. Two heredity integrals are presented relating, first, the sliding mechanics and oxide film repassivation physics to the current, and second, relating the electrode potential to the current using impedance concepts. Current-potential-time responses to controlled fretting conditions were measured across a fretting frequency from 0.2 to 10 Hz and compared to theoretical results. The coupled integrals were shown to predict the overall current-potential-time behavior for CoCrMo alloy surfaces under several controlled fretting corrosion conditions (loads, sliding speeds, etc.) with a high degree of similarity. These models can be adapted to numerical analyses of tribocorrosion to predict performance.

Chemical shift tensors obtained from solid-state NMR spectroscopy are very sensitive reporters of structure and dynamics in proteins. While accurate (13)C and (15)N chemical shift tensors are accessible by magic angle spinning (MAS) NMR, their quantum mechanical calculations remain challenging, particularly for (15)N atoms. Here we compare experimentally determined backbone (13)C(α) and (15)N(H) chemical shift tensors by MAS NMR with hybrid quantum mechanics/molecular mechanics/molecular dynamics (MD-QM/MM) calculations for the carbohydrate-binding domain of galectin-3. Excellent agreement between experimental and computed (15)N(H) chemical shift anisotropy values was obtained using the Amber ff15ipq force field when solvent dynamics was taken into account in the calculation. Our results establish important benchmark conditions for improving the accuracy of chemical shift calculations in proteins and may aid in the validation of protein structure models derived by MAS NMR.

The dinuclear copper enzyme, tyrosinase, activates O2 to form a (μ-η2 :η2 -peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X-ray crystal structures of the active tyrosinases with substrate l-tyrosine. At their catalytic sites, CuA moved toward l-tyrosine (CuA1 → CuA2), whose phenol oxygen directly coordinates to CuA2, involving the movement of CuB (CuB1 → CuB2). The crystal structures and spectroscopic analyses of the dioxygen-bound tyrosinases demonstrated that the peroxide ligand rotated, spontaneously weakening its O-O bond. Thus, the copper migration induced by the substrate-binding is accompanied by rearrangement of the bound peroxide species so as to provide one of the peroxide oxygen atoms with access to the phenol substrate's ϵ carbon atom.

Despite the significant influence of solution temperature on the structure of electrical double layer, the lack of theoretical model intercepts us to explain and predict the interesting experimental observations. In this work, we study the structure of electrical double layer as a function of thermochemical properties of the solution by proposing a phenomenological temperature dependent surface complexation model. We found that by introducing a buffer layer between the diffuse layer and stern layer, one can explain the sensitivity of zeta potential to temperature for different bulk ion concentrations. Calculation of the electrical conductance as function of thermochemical properties of solution reveals the electrical conductance not only is a function of bulk ion concentration and channel height but also the solution temperature. The present work model can provide deep understanding of micro- and nanofluidic devices functionality at different temperatures.

Flavin‐dependent ‘ene’‐reductases (EREDs) are highly selective catalysts for the asymmetric reduction of activated alkenes. This function is, however, limited to enones, enoates, and nitroalkenes using the native hydride transfer mechanism. Here we demonstrate that EREDs can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. Experimental evidence suggests the reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to the corresponding neutral benzylic radical in solution. DFT calculations reveal this radical to be “dynamically stable”, suggesting it is sufficiently long lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer. This reduction mechanism is distinct from the native one, highlighting the opportunity to expand the synthetic capabilities of existing enzyme platforms by exploiting new mechanistic paradigms.

A complete mathematical model for electromigration in paper-based analytical devices is derived, based on differential equations describing the motion of fluids by pressure sources and EOF, the transport of charged chemical species, and the electric potential distribution. The porous medium created by the cellulose fibers is considered like a network of tortuous capillaries and represented by macroscopic parameters following an effective medium approach. The equations are obtained starting from their open-channel counterparts, applying scaling laws and, where necessary, including additional terms. With this approach, effective parameters are derived, describing diffusion, mobility, and conductivity for porous media. While the foundations of these phenomena can be found in previous reports, here, all the contributions are analyzed systematically and provided in a comprehensive way. Moreover, a novel electrophoretically driven dispersive transport mechanism in porous materials is proposed. Results of the numerical implementation of the mathematical model are compared with experimental data, showing good agreement and supporting the validity of the proposed model. Finally, the model succeeds in simulating a challenging case of free-flow electrophoresis in paper, involving capillary flow and electrophoretic transport developed in a 2D geometry. Fil: Schaumburg, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentina Fil: Berli, Claudio Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

In this paper, we provide a theory to quantify the ionic current ( iion ) in nanochannels grafted with pH-responsive polyelectrolyte (PE) brushes. We consider the PE brushes to be modeled by our recently proposed augmented strong stretching theory (SST) model that improves the existing SST models by incorporating the effects of excluded volume interactions and an extended mass action law. Use of such augmented SST for this problem implies that this is the first study on computing iion in PE brush-grafted nanochannels accounting for the appropriate coupled configuration-electrostatic description of the PE brushes. iion is obtained as functions of PE brush grafting density, medium pH and salt concentration ( c∞ ), and the density of polyelectrolyte chargeable sites (PECS). For large c∞ , iion increases linearly with c∞ (as for such c∞ , iion becomes independent of the PE charge and is dominated by the bulk mobility and number density of the electrolyte ions), whereas iion is independent of c∞ at small c∞ (where the electric double layer electrostatics and the total number of ions in the system is dominated by the hydrogen ions). We further witness an enhancement of iion for smaller pH and larger grafting density at low and moderate c∞ , while there is little to no effect of the PECS density on the ionic current except for weakly grafted brushes at low c∞ . We anticipate that this study will serve as a theoretical foundation for a large number of applications that are based on the brush-induced modification of the ionic current in a nanochannel.

To accurately predict the environmental fate of per- and polyfluoroalkyl substances (PFAS), high-quality physicochemical property data are required. Because such data are often not available from experiments, assessment of the accuracy of existing property estimation models is essential. The quality of predicted physicochemical property data for a set of 25 PFAS was examined using COSMOtherm, EPI Suite, the estimation models accessible through the US Environmental Protection Agency's CompTox Chemicals Dashboard, and Linear Solvation Energy Relationships (LSERs) available through the UFZ-LSER Database. The results showed that COSMOtherm made the most accurate acid dissociation constant and air-water partition ratio estimates compared with literature data. The OPEn structure-activity/property Relationship App (OPERA; developed through the CompTox Chemicals Dashboard) estimates of vapor pressure and dry octanol-air partition ratios were the most accurate compared with other models of interest. Wet octanol-water partition ratios were comparably predicted by OPERA and EPI Suite, and the organic carbon soil coefficient and solubility were well predicted by OPERA and COSMOtherm. Acid dissociation of the perfluoroalkyl acids has a significant impact on their physicochemical properties, and corrections for ionization were included where applicable. Environ Toxicol Chem 2020;39:775-786. © 2020 SETAC.

Screened repulsion between uniformly charged plates with an intervening electrolyte is analyzed for strongly overlapped electrical double layers (EDL), accounting for the steric effect of ions and their expulsion from EDL edges into the surrounding solution. As a generalization of a study by Philipse et al. which does not account for these effects, an analytical expression is derived for the repulsion pressure in the limit of infinitely long plates with a zero-field assumption, which agrees closely with the corresponding numerical solution at low inter-plate separations. Our results show an augmented repulsive pressure for finite-sized ions at strong EDL overlaps. For plates with a finite lateral size, we demonstrate a further extended domain of low inter-plate gaps where the repulsion pressure increases with ion size due to a strong interplay between the steric interaction of ions and the EDL overspill phenomenon, considered earlier in a study by Ghosal & Sherwood limited to the linear Debye-Huckel regime (which cannot account for the steric effect of ions). This investigation on a simple model should enhance our understanding of the interaction between charged particles in electrophoresis, nanoscale self-assembly, active particles, and various other electrokinetic systems.

Vortex formation near a two-part cylinder with zeta potentials of different values but the same sign under an external DC electric field is numerically investigated in this paper. The cylinder, inserted in a straight microchannel filled with an aqueous solution, is composed of an upstream part and a downstream part. When a DC electric field is applied in the channel, under certain conditions, the vortex will form near the cylinder due to the different velocities of electroosmotic flow generated on the cylinder surface. The numerical results reveal that the larger the velocity difference of electroosmotic flow generated on the two-part cylinder and the smaller the channel width, the more conducive to vortex formation in the channel. In addition, if the zeta potential ratios of cylinder downstream part to upstream part and channel wall to cylinder upstream part are unchanged, the DC electric field strength and the zeta potential value do not affect the pattern of vortices formed in the channel. This study provides a way for vortex formation in microchannels and has the potential application in microfluidic devices.

In this paper, an investigation of the electroosmotic flow of fractional Oldroyd-B fluids in a narrow circular tube with high zeta potential is presented. The Navier linear slip law at the walls is considered. The potential field is applied along the walls described by the nonlinear Poisson-Boltzmann equation. It's worth noting here that the linear Debye-Hückel approximation can't be used at the condition of high zeta potential and the exact solution of potential in cylindrical coordinates can't be obtained. Therefore, the Matlab bvp4c solver method and the finite difference method are employed to numerically solve the nonlinear Poisson-Boltzmann equation and the governing equations of the velocity distribution, respectively. To verify the validity of our numerical approach, a comparison has been made with the previous work in the case of low zeta potential and the excellent agreement between the solutions is clear. Then, in view of the obtained numerical solution for the velocity distribution, the numerical solutions of the flow rate and the shear stress are derived. Furthermore, based on numerical analysis, the influence of pertinent parameters on the potential distribution and the generation of flow is presented graphically.

We provide an experimental and computational framework for (2)H quadrupolar chemical exchange saturation transfer NMR experiments (Q-CEST) under static solid-state conditions for the quantification of dynamics on μs-ms timescales. Simulations using simple 2-site exchange models provide insights into the relation between spin dynamics and motions. Biological applications focus on two sites of amyloid-β fibrils in the 3-fold symmetric polymorph. The first site, the methyl group of A2 of the disordered N-terminal domain, undergoes diffusive motions and conformational exchange due to transient interactions. Earlier (2)H rotating frame relaxation and quadrupolar CPMG measurements are combined with the Q-CEST approach to characterize the multiple conformational states of the domain. The second site, the methyl group of M35, spans the water-accessible cavity inside the fibrils’ core and undergoes extensive rotameric exchange. Q-CEST permits us to refine the rotameric exchange model for this site and allows the more precise determination of populations and rotameric exchange rate constants than line shape analysis.

We have 3D printed and fabricated micro free-flow electrophoresis (μFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. Two dimensional, nano liquid chromatography–micro free flow electrophoresis (2D nLC×μFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein. Surface adsorption was observed in separations of Chromeo P503 labeled myoglobin and cytochrome c but was significantly reduced compared to previously reported glass devices. Peak widths of 20 min. A 2D nLC×μFFE separation of a Chromeo P503 labeled tryptic digest of bovine serum albumin (BSA) was performed to demonstrate the high peak capacity possible due to the low surface adsorption in the 3D printed ABS devices, even in the absence of surface coatings or buffer additives.

Electroosmotic flow (EOF) has been widely used to transport fluids and samples in micro- and nanofluidic channels for lab-on-a-chip applications. This essentially surface-driven plug-like flow is, however, sensitive to both the fluid and wall properties, of which any inhomogeneity may draw disturbances to the flow and even instabilities. Existing studies on EOF instabilities have been focused primarily upon Newtonian fluids though many of the chemical and biological solutions are actually non-Newtonian. We carry out a systematic experimental investigation of the fluid rheological effects on the elastic instability in the EOF of phosphate buffer-based polymer solutions through T-shaped microchannels. We find that electro-elastic instabilities can be induced in shear thinning polyacrylamide (PAA) and xanthan gum (XG) solutions if the applied direct current voltage is above a threshold value. However, no instabilities are observed in Newtonian or weakly shear thinning viscoelastic fluids including polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), and hyaluronic acid (HA) solutions. We also perform a quantitative analysis of the wave parameters for the observed elasto-elastic instabilities.

Iridium(III) complexes have attracted more and more attention in the past few years because of their potential antineoplastic activity. In this study, four IrIII complexes of the types [(η5 -Cpx )Ir(N^N)Cl]PF6 (complexes 1 and 2) and [Ir(Phpy)2 (N^N)]PF6 (complexes 3 and 4) have been synthesized and characterized. They exhibit potential antineoplastic activity towards A549 cells, especially in the case of complex 1 [IC50 =(3.56±0.5) μm], which was nearly six times as effective as cisplatin [(21.31±1.7) μm]. Additionally, these complexes show some selectivity towards cancer cells over normal cells. They could be transported by serum albumin (binding constants were changed from 0.37×105 to 81.71×105 m-1 ). IrIII complexes 1 and 2 could catalyze the transformation of nicotinamide adenine dinucleotide reduced form (NADH) into NAD+ (turnover numbers 43.2, 11.9] and induce the accumulation of reactive oxygen species, thus confirming their antineoplastic mechanism of oxidation, whereas the cyclometalated complexes 3 and 4 were able to target the lysosome [Pearson co-localization coefficient (PCC)=0.73], cause lysosomal damage, and induce apoptosis. Understanding the mechanism of action would help further structure-activity optimization on these IrIII complexes as emerging cancer therapeutics.

Moment equations were developed on the basis of the Einstein equation for diffusion and the random walk model to analytically determine the rate constant for the interfacial solute permeation from a bulk solvent into molecular aggregates (kin ) and the inverse rate constant from the molecular aggregates to the bulk solvent (kout ). The moment equations were in good agreement with those derived in a different manner. To demonstrate their effectiveness in one concrete example, the moment equations were used to analytically determine the values of kin and kout of three electrically neutral solutes, i.e. resorcinol, phenol, and nitrobenzene, from the first absolute (μ1A ) and second central (μ2C ) moments of their elution peaks, as measured by electrokinetic chromatography (EKC), in which the sodium dodecyl sulfate (SDS) micelles were used as a pseudostationary phase. The values of kin and kout should be determined with no chemical modifications and no physical action with the molecular aggregates because they are dynamic systems formed through weak interactions between the components. The moment analysis of the elution peak profiles measured by EKC is effective to unambiguously determine kin , kout , and the partition equilibrium constant (kin /kout ) under appropriate experimental conditions.

Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-d-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far.

This is the peer reviewed version of the following article: Feehan, J.F., Monaghan, J., Gill, C.G., & Krogh, E.T. (2019). Direct measurement of acid dissociation constants of trace organic compounds at nanomolar levels in aqueous solution by condensed phase membrane introduction mass spectrometry. Environmental Toxicology and Chemistry, 38(9), 1879-1889. DOI: 10.1002/etc.4519, which has been published in final form at https://doi.org/10.1002/etc.4519. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. We report the use of condensed phase membrane introduction mass spectrometry as a novel method for the determination of acid dissociation constants for hydrophobic organic acids in aqueous solution at nanomolar concentrations. The technique is based on the pH dependent permeation of analytes through a semi-permeable polydimethylsiloxane membrane probe that is immersed directly in aqueous samples. We describe the method and report pKa values for compounds of biological and environmental relevance, including contaminants, pharmaceuticals and naphthenic acids. The approach can be applied to individual compounds, combined suites and complex mixtures at parts-per-billion levels. We report pKa values for ten carboxylic acids with precision estimates and relative errors (where reliable literature values are available) of less than 0.1 log units. Acidity constants for 2-methyl-3-methoxy-4-phenyl butanoic acid (a biomarker for microcystin algal toxins) and 4-t-butylcyclohexane carboxylic acid (a model naphthenic acid) are reported here as 4.28±0.03 and 5.15±0.05, respectively. Further, we employ this approach to measure the effect of both temperature and deuterium oxide (heavy water) on acid dissociation, reporting the enthalpy and entropy changes for the ionization of a representative carboxylic acid and substituted phenol. Natural Sciences and Engineering Research Council of Canada. Grant Number: Discovery Grant 2016‐06454 Post-print version This is the peer reviewed version of the following article: Feehan, J.F., Monaghan, J., Gill, C.G., & Krogh, E.T. (2019). Direct measurement of acid dissociation constants of trace organic compounds at nanomolar levels in aqueous solution by condensed phase membrane introduction mass spectrometry. Environmental Toxicology and Chemistry, 38(9), 1879-1889. DOI: 10.1002/etc.4519, which has been published in final form at https://doi.org/10.1002/etc.4519. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. https:/viurrspace.ca/bitstream/handle/10613/23324/GillKroghETC.pdf?sequence=3

An ever‐increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so‐called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined—Lego like—than in proteins, hence the numerous revelations of RNA excited states., RNA is a flexible molecule: This is what makes it functional, but also difficult to study, especially when the goal is to access those dynamic, often low‐populated and short‐lived conformers. Here the NMR methods and the structural insights, which have so far been obtained for RNAs and their picosecond‐to‐hour timescales are reviewed.