Effect of Solvent Properties on the Critical Solution Temperature of Thermoresponsive Polymers.

The ability of thermoresponsive polymers to respond to temperature with a reversible conformational change makes them promising 'smart' materials for solutions in medical and biotechnological applications. In this work, two such polymers and structural isomers were studied: poly(N-isopropyl acrylamide) (PNiPAm) and poly(2-isopropyl-2-oxazoline) (PiPOx). We compare the critical solution temperatures (CST) of these polymers in D2O and H2O in the presence of Hofmeister series salts, as results obtained under these different solvent conditions are often compared. D2O has a higher dipole moment and electronegativity than H2O, which could significantly alter the CST transition. We used two complementary methods to measure the CST, dynamic light scattering (DLS) and differential scanning calorimetry (DSC) and found that the CST decreased significantly in D2O compared to H2O. In the presence of highly concentrated kosmotropes, the CST of both polymers decreased in both solvents. The influence of the kosmotropic anions was smaller than the water isotope effect at low ionic strengths but considerably higher at physiological ionic strengths. However, the Hofmeister anion effect was quantitatively different in H2O than in D2O, with the largest relative differences observed for Cl-, where the CSTs in D2O decreased more than in H2O measured by DLS but less by DSC. PiPOx was more sensitive than PNiPAm to the presence of chaotropes. It exhibited much higher transition enthalpies and multistep transitions, especially in aqueous solutions. Our results highlight that measurements of thermoresponsive polymer properties in D2O cannot be compared directly or quantitatively to application conditions or even measurements performed in H2O.

NMR Dynamic View of the Stabilization of the WW4 Domain by Neutral NaCl and Kosmotropic Na2SO4 and NaH2PO4.

The Hofmeister series categorizes ions based on their effects on protein stability, yet the microscopic mechanism remains a mystery. In this series, NaCl is neutral, Na2SO4 and Na2HPO4 are kosmotropic, while GdmCl and NaSCN are chaotropic. This study employs CD and NMR to investigate the effects of NaCl, Na2SO4, and Na2HPO4 on the conformation, stability, binding, and backbone dynamics (ps-ns and µs-ms time scales) of the WW4 domain with a high stability and accessible side chains at concentrations ≤ 200 mM. The results indicated that none of the three salts altered the conformation of WW4 or showed significant binding to the four aliphatic hydrophobic side chains. NaCl had no effect on its thermal stability, while Na2SO4 and Na2HPO4 enhanced the stability by ~5 °C. Interestingly, NaCl only weakly interacted with the Arg27 amide proton, whereas Na2SO4 bound to Arg27 and Phe31 amide protons with Kd of 32.7 and 41.6 mM, respectively. Na2HPO4, however, bound in a non-saturable manner to Trp9, His24, and Asn36 amide protons. While the three salts had negligible effects on ps-ns backbone dynamics, NaCl and Na2SO4 displayed no effect while Na2HPO4 significantly increased the µs-ms backbone dynamics. These findings, combined with our recent results with GdmCl and NaSCN, suggest a microscopic mechanism for the Hofmeister series. Additionally, the data revealed a lack of simple correlation between thermodynamic stability and backbone dynamics, most likely due to enthalpy-entropy compensation. Our study rationalizes the selection of chloride and phosphate as the primary anions in extracellular and intracellular spaces, as well as polyphosphate as a primitive chaperone in certain single-cell organisms.

NMR Dynamic View of the Destabilization of WW4 Domain by Chaotropic GdmCl and NaSCN.

GdmCl and NaSCN are two strong chaotropic salts commonly used in protein folding and stability studies, but their microscopic mechanisms remain enigmatic. Here, by CD and NMR, we investigated their effects on conformations, stability, binding and backbone dynamics on ps-ns and µs-ms time scales of a 39-residue but well-folded WW4 domain at salt concentrations ≤200 mM. Up to 200 mM, both denaturants did not alter the tertiary packing of WW4, but GdmCl exerted more severe destabilization than NaSCN. Intriguingly, GdmCl had only weak binding to amide protons, while NaSCN showed extensive binding to both hydrophobic side chains and amide protons. Neither denaturant significantly affected the overall ps-ns backbone dynamics, but they distinctively altered µs-ms backbone dynamics. This study unveils that GdmCl and NaSCN destabilize a protein before the global unfolding occurs with differential binding properties and µs-ms backbone dynamics, implying the absence of a simple correlation between thermodynamic stability and backbone dynamics of WW4 at both ps-ns and µs-ms time scales.

Ion-Specific Interactions Engender Dynamic and Tailorable Properties in Biomimetic Cationic Polyelectrolytes.

Biomaterials such as spider silk and mussel byssi are fabricated by the dynamic manipulation of intra- and intermolecular biopolymer interactions. Organisms modulate solution parameters, such as pH and ion co-solute concentration, to effect these processes. These biofabrication schemes provide a conceptual framework to develop new dynamic and responsive abiotic soft material systems. Towards these ends, the chemical diversity of readily available ionic compounds offers a broad palette to manipulate the physicochemical properties of polyelectrolytes via ion-specific interactions. In this study, we show for the first time that the ion-specific interactions of biomimetic polyelectrolytes engenders a variety of phase separation behaviors, creating dynamic, thermal and ion responsive soft matter. that exhibits a spectrum of physical properties, spanning viscous fluids, to viscoelastic and viscoplastic solids. These ion dependent characteristics are further rendered general by the merger of lysine and phenylalanine into a single, amphiphilic vinyl monomer. The unprecedented breadth, precision, and dynamicity in the reported ion dependent phase behaviors thus introduce a broad array of opportunities for the future development of responsive soft matter, properties that are poised to drive developments in critical areas such as chemical sensing, soft robotics, and additive manufacturing.

Solvation of Nanoions in Aqueous Solutions.

In recent years it has been increasingly recognized that different classes of large ions with multiple valency have effects conceptually similar to weakly solvated ions in the Hofmeister series, also labeled by the term chaotropic. The term "superchaotropic effect" has been coined because these effects are much more strongly pronounced for nanometer-sized ions, whose adsorption properties often resemble typical surfactants. Despite this growing interest in these nanometer-sized ions, a simple conceptual extension of the Hofmeister series toward nanoions has not been achieved because an extrapolation of the one-dimensional surface charge density scale does not lead to the superchaotropic regime. In this work, we discuss a generic model that is broadly applicable to ions of nearly spherical shape and thus includes polyoxometalates and boron clusters. We present a qualitative classification scheme in which the ion size appears as a second dimension. Ions of different sizes but the same charge density differ in their bulk solvation free energy. As the ions grow bigger at constant surface charge density, they become more stable in solution, but the adsorption behavior is still governed by the surface charge density. A detailed molecular dynamics simulation study of large ions that is based on a shifted Lennard-Jones potential is presented that supports the presented classification scheme.

Hofmeister Series: From Aqueous Solution of Biomolecules to Single Cells and Nanovesicles.

Hofmeister effects play a critical role in numerous physicochemical and biological phenomena, including the solubility and/or accumulation of proteins, the activities of enzymes, ion transport in biochannels, the structure of lipid bilayers, and the dynamics of vesicle opening and exocytosis. This minireview focuses on how ionic specificity affects the physicochemical properties of biomolecules to regulate cellular exocytosis, vesicular content, and nanovesicle opening. We summarize recent progress in further understanding Hofmeister effects on biomacromolecules and their applications in biological systems. These important steps have increased our understanding of the Hofmeister effects on cellular exocytosis, vesicular content, and nanovesicle opening. Increasing evidence is firmly establishing that the ions along the Hofmeister series play an important role in living organisms that has often been ignored.

Electrical Properties of Taste Sensors with Positively Charged Lipid Membranes Composed of Amines and Ammonium Salts.

Currently, taste sensors utilizing lipid polymer membranes are utilized to assess the taste of food products quantitatively. During this process, it is crucial to identify and quantify basic tastes, e.g., sourness and sweetness, while ensuring that there is no response to tasteless substances. For instance, suppression of responses to anions, like tasteless NO3- ions contained in vegetables, is essential. However, systematic electrochemical investigations have not been made to achieve this goal. In this study, we fabricated three positively charged lipid polymer membranes containing oleylamine (OAm), trioctylemethylammonium chloride (TOMACl), or tetradodecylammonium bromide (TDAB) as lipids, and sensors that consist of these membranes to investigate the potential change characteristics of these sensors in solutions containing different anions (F-, Cl-, Br-, NO3-, I-). The ability of each anion solution to reduce the positive charge on membranes and shift the membrane potential in the negative direction was in the following order: I- > NO3- > Br- > Cl- > F-. This order well reflected the order of size of the hydrated ions, related to their hydration energy. Additionally, the OAm sensor displayed low ion selectivity, whereas the TOMACl and TDAB sensors showed high ion selectivity related to the OAm sensor. Such features in ion selectivity are suggested to be due to the variation in positive charge with the pH of the environment and packing density of the OAm molecule in the case of the OAm sensor and due to the strong and constant positive charge created by complete ionization of lipids in the case of TOMACl and TDAB sensors. Furthermore, it was revealed that the ion selectivity varies by changing the lipid concentration in each membrane. These results contribute to developing sensor membranes that respond to different anion species selectively and creating taste sensors capable of suppressing responses to tasteless anions.

Modulation of Insulin Amyloid Fibrillization in Imidazolium-Based Ionic Liquids with Hofmeister Series Anions.

Amyloid fibrils have immense potential to become the basis of modern biomaterials. The formation of amyloid fibrils in vitro strongly depends on the solvent properties. Ionic liquids (ILs), alternative solvents with tunable properties, have been shown to modulate amyloid fibrillization. In this work, we studied the impact of five ILs with 1-ethyl-3-methylimidazolium cation [EMIM+] and anions of Hofmeisterseries hydrogen sulfate [HSO4-], acetate [AC-], chloride [Cl-], nitrate [NO3-], and tetrafluoroborate [BF4-] on the kinetics of insulin fibrillization and morphology, and the structure of insulin fibrils when applying fluorescence spectroscopy, AFM and ATR-FTIR spectroscopy. We found that the studied ILs were able to speed up the fibrillization process in an anion- and IL-concentration-dependent manner. At an IL concentration of 100 mM, the efficiency of the anions at promoting insulin amyloid fibrillization followed the reverse Hofmeister series, indicating the direct binding of ions with the protein surface. At a concentration of 25 mM, fibrils with different morphologies were formed, yet with similar secondary structure content. Moreover, no correlation with the Hofmeister ranking was detected for kinetics parameters. IL with the kosmotropic strongly hydrated [HSO4-] anion induced the formation of large amyloid fibril clusters, while the other kosmotropic anion [AC-] along with [Cl-] led to the formation of fibrils with similar needle-like morphologies to those formed in the IL-free solvent. The presence of the ILs with the chaotropic anions [NO3-] and [BF4-] resulted in longer laterally associated fibrils. The effect of the selected ILs was driven by a sensitive balance and interplay between specific protein-ion and ion-water interactions and non-specific long-range electrostatic shielding.

Beta-Barrel Channel Response to High Electric Fields: Functional Gating or Reversible Denaturation?

Ion channels exhibit gating behavior, fluctuating between open and closed states, with the transmembrane voltage serving as one of the essential regulators of this process. Voltage gating is a fundamental functional aspect underlying the regulation of ion-selective, mostly α-helical, channels primarily found in excitable cell membranes. In contrast, there exists another group of larger, and less selective, ß-barrel channels of a different origin, which are not directly associated with cell excitability. Remarkably, these channels can also undergo closing, or "gating", induced by sufficiently strong electric fields. Once the field is removed, the channels reopen, preserving a memory of the gating process. In this study, we explored the hypothesis that the voltage-induced closure of the ß-barrel channels can be seen as a form of reversible protein denaturation by the high electric fields applied in model membranes experiments-typically exceeding twenty million volts per meter-rather than a manifestation of functional gating. Here, we focused on the bacterial outer membrane channel OmpF reconstituted into planar lipid bilayers and analyzed various characteristics of the closing-opening process that support this idea. Specifically, we considered the nearly symmetric response to voltages of both polarities, the presence of multiple closed states, the stabilization of the open conformation in channel clusters, the long-term gating memory, and the Hofmeister effects in closing kinetics. Furthermore, we contemplate the evolutionary aspect of the phenomenon, proposing that the field-induced denaturation of membrane proteins might have served as a starting point for their development into amazing molecular machines such as voltage-gated channels of nerve and muscle cells.

Effects of different physical factors on potato protein-chitosan complexes considering Hofmeister series and Maillard reaction.

BACKGROUND: Potato protein possesses strong potential for application in the food industry due to its outstanding nutritional and functional properties. However, the inevitable industrial processing often brings adverse effects. The use of a polysaccharide and protein complex is a promising way to improve the performance of potato protein. This work aimed to investigate the effects of different physical factors on the potato protein/chitosan (PP/CS) complex system. RESULTS: The addition of NaCl was not conductive to the formation of PP/CS complexes, resulting in significantly decreased peak turbidities from 1.29 to 0.75. The effect of different ions on PP/CS system matched with the Hofmeister series in the following order: Li+ > Control > Na+ > K+ ; SCN- > I- > NO3 - > Br- ≈ Control > Cl- > SO4 2- , among which the salting-in ions (Li+ , Br- , NO3 - , I- and SCN- ) tended to promote the formation of PP/CS complexes. The turbidity increased significantly when the reaction temperature rose to 45 °C and above, and peak turbidity was obtained at lower pH values. The PP/CS system reaction at 45 °C led to the highest whiteness value, and the Maillard reaction could occur when the temperature was above 45 °C. CONCLUSIONS: The results of the present study confirmed that different physical factors led to strong influences on PP/CS complexes, especially when considering the Hofmeister series and the Maillard reaction. These findings could have significant implications for the utilization of potato protein in complex food systems. © 2023 Society of Chemical Industry.

The murburn precepts for cellular ionic homeostasis and electrophysiology.

Starting from the basic molecular structure and redox properties of its components, we build a macroscopic cellular electrophysiological model. We first present a murburn purview that could explain ion distribution in bulk-milieu/membrane-interface and support the origin of trans-membrane potential (TMP) in cells. In particular, the discussion focuses on how cells achieve disparity in the distribution of monovalent and divalent cations within (K+ > Na+ > Mg2+ > Ca2+ ) and outside (Na+ > K+ > Ca2+ > Mg2+ ). We explore how TMP could vary for resting/graded/action potentials generation and project a model for impulse conduction in neurons. Outcomes based on murburn bioenergetic equilibriums leading to solubilization of ion-pairs, membrane's permittivity, protein channels' fluxes, and proteins' innate ability to bind/adsorb ions selectively are projected as the integral rationale. We also provide experimental modalities to ratify the projections.

Million-fold sensitivity enhancement in proteopathic seed amplification assays for biospecimens by Hofmeister ion comparisons.

Recent work with prion diseases and synucleinopathies indicates that accurate diagnostic methods for protein-folding diseases can be based on the ultrasensitive, amplified measurement of pathological aggregates in biospecimens. A better understanding of the physicochemical factors that control the seeded polymerization of such aggregates, and their amplification in vitro, should allow improvements in existing assay platforms, as well as the development of new assays for other proteopathic aggregates. Here, we systematically investigated the effects of the ionic environment on the polymerization of tau, α-synuclein, and the prion protein (PrP) induced by aggregates in biospecimens. We screened salts of the Hofmeister series, a relative ordering of strongly and weakly hydrated salts that tend to precipitate or solubilize proteins. We found that sensitivities of tau-based assays for Alzheimer's seeds and PrP-based assays for prions were best in weakly hydrated anions. In contrast, we saw an inverse trend with different tau-based assays, improving detection sensitivity for progressive supranuclear palsy seeds by ≈106 Hofmeister analysis also improved detection of sporadic Creutzfeldt-Jakob disease prions in human nasal brushings and chronic wasting disease prions in deer-ear homogenates. Our results demonstrate strong and divergent influences of ionic environments on the amplification and detection of proteopathic seeds as biomarkers for protein-folding diseases.

The Lyotropic Nature of Halates: An Experimental Study.

Unlike halides, where the kosmotropicity decreases from fluoride to iodide, the kosmotropic nature of halates apparently increases from chlorate to iodate, in spite of the lowering in the static ionic polarizability. In this paper, we present an experimental study that confirms the results of previous simulations. The lyotropic nature of aqueous solutions of sodium halates, i.e., NaClO3, NaBrO3, and NaIO3, is investigated through density, conductivity, viscosity, and refractive index measurements as a function of temperature and salt concentration. From the experimental data, we evaluate the activity coefficients and the salt polarizability and assess the anions' nature in terms of kosmotropicity/chaotropicity. The results clearly indicate that iodate behaves as a kosmotrope, while chlorate is a chaotrope, and bromate shows an intermediate nature. This experimental study confirms that, in the case of halates XO3-, the kosmotropic-chaotropic ranking reverses with respect to halides. We also discuss and revisit the role of the anion's polarizability in the interpretation of Hofmeister phenomena.

Histidine-Mediated Ion Specific Effects Enable Salt Tolerance of a Pore-Forming Marine Antimicrobial Peptide.

Antimicrobial peptides (AMPs) preferentially permeate prokaryotic membranes via electrostatic binding and membrane remodeling. Such action is drastically suppressed by high salt due to increased electrostatic screening, thus it is puzzling how marine AMPs can possibly work. We examine as a model system, piscidin-1, a histidine-rich marine AMP, and show that ion-histidine interactions play unanticipated roles in membrane remodeling at high salt: Histidines can simultaneously hydrogen-bond to a phosphate and coordinate with an alkali metal ion to neutralize phosphate charge, thereby facilitating multidentate bonds to lipid headgroups in order to generate saddle-splay curvature, a prerequisite to pore formation. A comparison among Na+ , K+ , and Cs+ indicates that histidine-mediated salt tolerance is ion specific. We conclude that histidine plays a unique role in enabling protein/peptide-membrane interactions that occur in marine or other high-salt environment.

Simultaneous Counting of Molecules in the Halo and Dense-Core of Nanovesicles by Regulating Dynamics of Vesicle Opening.

We report the discovery that in the presence of chaotropic anions (SCN- ) the opening of nanometer biological vesicles at an electrified interface often becomes a two-step process (around 30 % doublet peaks). We have then used this to independently count molecules in each subvesicular compartment, the halo and protein dense-core, and the fraction of catecholamine binding to the dense-core is 68 %. Moreover, we differentiated two distinct populations of large dense-core vesicles (LDCVs) and quantified their content, which might correspond to immature (43 %) and mature (30 %) LDCVs, to reveal differences in their biogenesis. We speculate this is caused by an increase in the electrostatic attraction between protonated catecholamine and the negatively charged dense-core following adsorption of SCN- .

Specific Ion-Protein Interactions Influence Bacterial Ice Nucleation.

Ice nucleation-active bacteria are the most efficient ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C, thereby overcoming the kinetically hindered phase transition process at these conditions. Using highly specialized ice-nucleating proteins (INPs), they can cause frost damage to plants and influence the formation of clouds and precipitation in the atmosphere. In nature, the bacteria are usually found in aqueous environments containing ions. The impact of ions on bacterial ice nucleation efficiency, however, has remained elusive. Here, we demonstrate that ions can profoundly influence the efficiency of bacterial ice nucleators in a manner that follows the Hofmeister series. Weakly hydrated ions inhibit bacterial ice nucleation whereas strongly hydrated ions apparently facilitate ice nucleation. Surface-specific sum-frequency generation spectroscopy and molecular dynamics simulations reveal that the different effects are due to specific interactions of the ions with the INPs on the surface of the bacteria. Our results demonstrate that heterogeneous ice nucleation facilitated by bacteria strongly depends upon the nature of the ions, and specific ion-protein interactions are essential for the complete description of heterogeneous ice nucleation by bacteria.

Specific Ion Effects of Azobenzene Salts on Photoresponse of PNIPAm in Aqueous Solutions.

Ionic species are important to dominate phase separation behaviors of poly(N-isopropylacrylamide) (PNIPAm) in aqueous solutions. Herein, photoresponsive azobenzene-based salts with various ions are prepared and their photoresponsive ion effects on clouding temperatures (TcpS ) of PNIPAm in aqueous solutions are explored. It is found that, despite of various structures of anions and cations, trans-TcpS under vis light irradiation are always higher than cis-TcpS under UV irradiation. Particularly, Hofmeister effect of anions on TcpS is roughly observed. For example, azobenzene with kosmotropic CO3 2- gives the lowest cis-Tcp while in use of typical chaotropic anions, such as ClO4 - , azobenzene isomerization less affects values of Tcp s. In another hand, azobenzene-based metallic salts containing lithium, sodium, and potassium cations also demonstrate photoresponsive Hofmeister effect. Trans-metallic azobenzene demonstrates a chaotropic effect on Tcp s while UV induces kosmotropic behaviors on TcpS . Additionally, ionic conduction of the solution along with photoresponsive phase separations is also investigated and PNIPAm aggregations induce a sharp reduction of ion conduction during UV light illumination.

Electrical Properties of Two Types of Membrane Component Used in Taste Sensors.

The lipid phosphoric acid di-n-decyl ester (PADE) has played an important role in the development of taste sensors. As previously reported, however, the concentration of PADE and pH of the solution affected the dissociation of H+, which made the measurement results less accurate and stable. In addition, PADE caused deterioration in the response to bitterness because PADE created the acidic environment in the membrane. To solve these problems, our past study tried to replace the PADE with a completely dissociated substance called tetrakis [3,5-bis (trifluoromethyl) phenyl] borate sodium salt dehydrate (TFPB) as lipid. To find out whether the two substances can be effectively replaced, it is necessary to perform an in-depth study on the properties of the two membranes themselves. In this study, we fabricated two types of membrane electrodes, based on PADE or TFPB, respectively, using 2-nitrophenyl octyl ether (NPOE) as a plasticizer. We measured the selectivity to cations such as Cs+, K+, Na+ and Li+, and also the membrane impedance of the membranes comprising PADE or TFPB of the different concentrations. As a result, we found that any concentration of PADE membranes always had low ion selectivity, while the ion selectivity of TFPB membranes was concentration-dependent, showing increasing ion selectivity with the TFPB concentrations. The ion selectivity order was Cs+>K+>Na+>Li+. The hydration of ions was considered to participate in this phenomenon. In addition, the membrane impedance decreased with increasing PADE and TFPB concentrations, while the magnitudes differed, implying that there is a difference in the dissociation of the two substances. The obtained results will contribute to the development of novel receptive membranes of taste sensors.

Effect of Hofmeister Ions on Transport Properties of Aqueous Solutions of Sodium Hyaluronate.

Tracer diffusion coefficients obtained from the Taylor dispersion technique at 25.0 °C were measured to study the influence of sodium, ammonium and magnesium salts at 0.01 and 0.1 mol dm-3 on the transport behavior of sodium hyaluronate (NaHy, 0.1%). The selection of these salts was based on their position in Hofmeister series, which describe the specific influence of different ions (cations and anions) on some physicochemical properties of a system that can be interpreted as a salting-in or salting-out effect. In our case, in general, an increase in the ionic strength (i.e., concentrations at 0.01 mol dm-3) led to a significant decrease in the limiting diffusion coefficient of the NaHy 0.1%, indicating, in those circumstances, the presence of salting-in effects. However, the opposite effect (salting-out) was verified with the increase in concentration of some salts, mainly for NH4SCN at 0.1 mol dm-3. In this particular salt, the cation is weakly hydrated and, consequently, its presence does not favor interactions between NaHy and water molecules, promoting, in those circumstances, less resistance to the movement of NaHy and thus to the increase of its diffusion (19%). These data, complemented by viscosity measurements, permit us to have a better understanding about the effect of these salts on the transport behaviour of NaHy.

Free Energies of Hydrated Halide Anions: High Through-Put Computations on Clusters to Treat Rough Energy-Landscapes.

With a longer-term goal of addressing the comparative behavior of the aqueous halides F-, Cl-, Br-, and I- on the basis of quasi-chemical theory (QCT), here we study structures and free energies of hydration clusters for those anions. We confirm that energetically optimal (H2O)nX clusters, with X = Cl-, Br-, and I-, exhibit surface hydration structures. Computed free energies, based on optimized surface hydration structures utilizing a harmonic approximation, typically (but not always) disagree with experimental free energies. To remedy the harmonic approximation, we utilize single-point electronic structure calculations on cluster geometries sampled from an AIMD (ab initio molecular dynamics) simulation stream. This rough-landscape procedure is broadly satisfactory and suggests unfavorable ligand crowding as the physical effect addressed. Nevertheless, this procedure can break down when n≳4, with the characteristic discrepancy resulting from a relaxed definition of clustering in the identification of (H2O)nX clusters, including ramified structures natural in physical cluster theories. With ramified structures, the central equation for the present rough-landscape approach can acquire some inconsistency. Extension of these physical cluster theories in the direction of QCT should remedy that issue, and should be the next step in this research direction.