Biomaterials Adapted to Vat Photopolymerization in 3D Printing: Characteristics and Medical Applications.

Along with the rapid and extensive advancements in the 3D printing field, a diverse range of uses for 3D printing have appeared in the spectrum of medical applications. Vat photopolymerization (VPP) stands out as one of the most extensively researched methods of 3D printing, with its main advantages being a high printing speed and the ability to produce high-resolution structures. A major challenge in using VPP 3D-printed materials in medicine is the general incompatibility of standard VPP resin mixtures with the requirements of biocompatibility and biofunctionality. Instead of developing completely new materials, an alternate approach to solving this problem involves adapting existing biomaterials. These materials are incompatible with VPP 3D printing in their pure form but can be adapted to the VPP chemistry and general process through the use of innovative mixtures and the addition of specific pre- and post-printing steps. This review's primary objective is to highlight biofunctional and biocompatible materials that have been adapted to VPP. We present and compare the suitability of these adapted materials to different medical applications and propose other biomaterials that could be further adapted to the VPP 3D printing process in order to fulfill patient-specific medical requirements.

Prokaryotic Na+/H+ Exchangers-Transport Mechanism and Essential Residues.

Na+/H+ exchangers are essential for Na+ and pH homeostasis in all organisms. Human Na+/H+ exchangers are of high medical interest, and insights into their structure and function are aided by the investigation of prokaryotic homologues. Most prokaryotic Na+/H+ exchangers belong to either the Cation/Proton Antiporter (CPA) superfamily, the Ion Transport (IT) superfamily, or the Na+-translocating Mrp transporter superfamily. Several structures have been solved so far for CPA and Mrp members, but none for the IT members. NhaA from E. coli has served as the prototype of Na+/H+ exchangers due to the high amount of structural and functional data available. Recent structures from other CPA exchangers, together with diverse functional information, have allowed elucidation of some common working principles shared by Na+/H+ exchangers from different families, such as the type of residues involved in the substrate binding and even a simple mechanism sufficient to explain the pH regulation in the CPA and IT superfamilies. Here, we review several aspects of prokaryotic Na+/H+ exchanger structure and function, discussing the similarities and differences between different transporters, with a focus on the CPA and IT exchangers. We also discuss the proposed transport mechanisms for Na+/H+ exchangers that explain their highly pH-regulated activity profile.

Mutation of two key aspartate residues alters stoichiometry of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae.

Bacterial NhaB Na+/H+ exchangers belonging to the Ion Transporter superfamily are poorly characterized in contrast to Na+/H+ exchangers of the Cation Proton Antiporter superfamily which have NhaA from Escherichia coli as a prominent member. For a more detailed understanding of the intricacies of the exchanger's transport mechanism, mutational studies are essential. Therefore, we mutated two protonatable residues present in the putative transmembrane region of NhaB from Klebsiella pneumoniae (KpNhaB), which could serve as substrate binding sites, Asp146 and Asp404, to either glutamate or alanine and analyzed transport function and stability of the mutants using electrophysiological and fluorimetric techniques. While mutation of either Asp residue to Glu only had slight to moderate effects on the transport activity of the exchanger, the mutations D404A and D146A, in particular, had more profound effects on the transport function. Furthermore, a double mutant, D146A/D404A, exhibited a remarkable behavior at alkaline pH, where recorded electrical currents changed polarity, showing steady-state transport with a stoichiometry of H+:Na+ < 1, as opposed to the H+:Na+ > 1 stoichiometry of the WT. Thus, we showed that Asp146 and Asp404 are part of the substrate binding site(s) of KpNhaB and engineered a Na+/H+ exchanger with a variable stoichiometry.

Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter.

Much of the research on Na+/H+ exchange has been done in prokaryotic models, mainly on the NhaA Na+/H+-exchanger from Escherichia coli (EcNhaA). Two conserved aspartate residues, Asp-163 and Asp-164, are essential for transport and are candidates for possible binding sites for the two H+ that are exchanged for one Na+ to make the overall transport process electrogenic. More recently, a proposed mechanism of transport for EcNhaA has suggested direct binding of one of the transported H+ to the conserved Lys-300 residue, a salt bridge partner of Asp-163. This contention is supported by a study reporting that substitution of the equivalent residue, Lys-305, of a related Na+/H+ antiporter, NapA from Thermus thermophilus, renders the transporter electroneutral. In this work, we sought to establish whether the Lys-300 residue and its partner Asp-163 are essential for the electrogenicity of EcNhaA. To that end, we replaced Lys-300 with Gln, either alone or together with the simultaneous substitution of Asp-163 with Asn, and characterized these transporter variants in electrophysiological experiments combined with H+ transport measurements and stability analysis. We found that K300Q EcNhaA can still support electrogenic Na+/H+ antiport in EcNhaA, but has reduced thermal stability. A parallel electrophysiological investigation of the K305Q variant of TtNapA revealed that it is also electrogenic. Furthermore, replacement of both salt bridge partners in the ion-binding site of EcNhaA produced an electrogenic variant (D163N/K300Q). Our findings indicate that alternative mechanisms sustain EcNhaA activity in the absence of canonical ion-binding residues and that the conserved lysines confer structural stability.

Competition is the basis of the transport mechanism of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae.

Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.

Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter.

Na+/H+ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na+, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na+/H+ exchanger family and a model system for all related Na+/H+ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H+ ions that NhaA exchanges for one Na+ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na+ and H+ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na+/H+ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.

Electrogenic Cation Binding in the Electroneutral Na+/H+ Antiporter of Pyrococcus abyssi.

Na+/H+ antiporters in the CPA1 branch of the cation proton antiporter family drive the electroneutral exchange of H+ against Na+ ions and ensure pH homeostasis in eukaryotic and prokaryotic organisms. Although their transport cycle is overall electroneutral, specific partial reactions are electrogenic. Here, we present an electrophysiological study of the PaNhaP Na+/H+ antiporter from Pyrococcus abyssi reconstituted into liposomes. Positive transient currents were recorded upon addition of Na+ to PaNhaP proteoliposomes, indicating a reaction where positive charge is rapidly displaced into the proteoliposomes with a rate constant of k >200 s-1 We attribute the recorded currents to an electrogenic reaction that includes Na+ binding and possibly occlusion. Subsequently, positive charge is transported out of the cell associated with H+ binding, so that the overall reaction is electroneutral. We show that the differences in pH profile and Na+ affinity of PaNhaP and the related MjNhaP1 from Methanocaldococcus jannaschii can be attributed to an additional negatively charged glutamate residue in PaNhaP. The results are discussed in the context of the physiological function of PaNhaP and other microbial Na+/H+ exchangers. We propose that both, electroneutral and electrogenic Na+/H+ antiporters, represent a carefully tuned self-regulatory system, which drives the cytoplasmic pH back to neutral after any deviation.

A universal mechanism for transport and regulation of CPA sodium proton exchangers.

Recent studies performed on a series of Na+/H+ exchangers have led us to postulate a general mechanism for Na+/H+ exchange in the monovalent cation/proton antiporter superfamily. This simple mechanism employs a single binding site for which both substrates compete. The developed kinetic model is self-regulatory, ensuring down-regulation of transport activity at extreme pH, and elegantly explains the pH-dependent activity of Na+/H+ exchangers. The mechanism was experimentally verified and shown to describe both electrogenic and electroneutral exchangers. Using a small number of parameters, exchanger activity can be modeled under different conditions, providing insights into the physiological role of Na+/H+ exchangers.

Species differences in bacterial NhaA Na+/H+ exchangers.

Bacteria have adapted their NhaA Na(+)/H(+) exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H(+) and Na(+). We show that H.pylori NhaA only has a small acidic shift of its pH-dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.

NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.

pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers.

Na(+)/H(+) exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na(+)/H(+) exchangers, where a single binding site is alternatively occupied by Na(+) or one or two H(+) ions. The proposed transport mechanism inherently down-regulates Na(+)/H(+) exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na(+)/H(+) exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na(+)/H(+) exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na(+)/H(+) exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na(+)/H(+) exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na(+)/H(+) exchangers.

Introduction to solid supported membrane based electrophysiology.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire. After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage. The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis. This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

HPLC separation of acetaminophen and its impurities using a mixed-mode reversed-phase/cation exchange stationary phase.

Determination of acetaminophen and its main impurities: 4-nitrophenol, 4'-chloroacetanilide, as well as 4-aminophenol and its degradation products, p-benzoquinone and hydroquinone has been developed and validated by a new high-performance liquid chromatography method. Chromatographic separation has been obtained on a Hypersil Duet C18/SCX column, using gradient elution, with a mixture of phosphate buffer (pH = 4.88) and methanol as a mobile phase. Analysis time did not exceed 14.5 min and good resolutions, peak shapes and asymmetries have resulted. The linearity of the method has been tested in the range of 5.0-60 µg/mL for acetaminophen and 0.5-6 µg/mL for the other compounds. The limits of detection and quantification have been also established to be lower than 0.1 µg/mL and 0.5 µg/mL, respectively. The method has been successfully applied for the analysis of commercial acetaminophen preparations.