Water-Activated Semiquinone Formation and Carboxylic Acid Dissociation in Melanin Revealed by Infrared Spectroscopy

Eumelanin is a widespread biomacromolecule pigment in the biosphere and has been widely investigated for numerous bioelectronics and energetic applications. Many of these applications depend on eumelanin’s ability to conduct proton current at various levels of hydration. The origin of this behavior is connected to a comproportionation reaction between oxidized and reduced monomer moieties and water. A hydration-dependent FTIR spectroscopic study on eumelanin is presented herein, which allows for the first time tracking the comproportionation reaction via the gradual increase of the overall aromaticity of melanin monomers in the course of hydration. We identified spectral features associated with the presence of specific “one and a half” C𝌁O bonds, typical for o-semiquinones. Signatures of semiquinone monomers with internal hydrogen bonds and that carboxylic groups, in contrast to semiquinones, begin to dissociate at the very beginning of melanin hydration were indicated. As such, we suggest a modification to the common hydration-dependent conductivity mechanism and propose that the conductivity at low hydration is dominated by carboxylic acid protons, whereas higher hydration levels manifest semiquinone protons.

Amantadine Variant - Aryl Conjugates that Inhibit Multiple Amantadine Resistant M2 Mutant Influenza A Viruses

One challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza A viruses, which comprise several strains with variable susceptibility to antiviral drugs. Viruses bearing the S31N mutant of the M2, such as the pandemic 2009 H1N1 and seasonal H3N2, as well as other mutants (L26F, V27A, A30T, G34E) are resistant to amantadine class of drugs. Here, we synthesized and tested many of the second generation amantadine - aryl conjugates, against the WT M2 and all the M2 amantadine resistant strains, i.e. L26F, V27A, S31N, A30T, G34E generated from WSN/33 (S31N) virus. We identified many compounds that are dual in vitro M2 WT and L26F virus inhibitors. Furthermore, few of them (21, 32, 33), having a rimantadine or diamantadine or 4-(1-adamantyl)aniline instead of amantadine in the conjugate, were in vitro inhibitors against M2 WT, L26F and S31N while one of them inhibited also the A30T virus. The electrophysiology (EP) experiments showed that these compounds blocked significantly M2 WT, L26F or even M2 V27A channels but not the M2 S31N. The observation that adamantane variants and derivatives inhibit multiple M2 mutant virus replication in cell culture, without blocking M2 channel-mediated proton current in EP is not uncommon, underlying a mechanism of antiviral activity that has not been identified.

Amantadine Variants Activity Against Multiple Influenza A Viruses

Future pandemic influenza necessitates the development of new drugs against the current circulating, amantadine and rimantadine drugs resistant, influenza A M2 S31N viruses. The possibility of an antigenic shift to M2 S31 necessitates ranking the biological activities of amantadine variants. Several amantadine variants have been tested by different laboratories, but various M2 wild type influenza A strains have been used with different sensitivity against amantadine and the unambiguous comparison between potencies is not straightforward. Here, we compared the anti-influenza activities of 57 synthetic amantadine variants against influenza A WSN/33 viruses with amantadine-sensitive M2 WT, with a range of over three digits providing a reference set of potencies for structure-activity relationships, and amantadine-resistant M2 S31N proteins (and observed no potent compounds). 17 compounds were selected and tested against M2 L26F, V27A, A30T, G34E viruses. We tested few reference compounds using electrophysiology and explored point mutations which both showed that M2 is the target of potent antiviral potency against the M2 WT, L26F, V27A viruses. Major findings are: (a) Several amantadine variants from Kolocouris group block only M2 WT and M2 L26F-mediated proton current and the corresponding viruses replication. (b) A compound from Vazquez’s group is a triple blocker of M2 WT, L26F, V27A channels and viruses replication. (c) A compound from Vazquez’s group blocks only M2 L26 channel and virus replication. (d) Several compounds from Kolocouris group have potent activity against several influenza A M2 WT and three M2 S31N viruses, eg. the pandemic A/H1N1/California/07/2009 (H1N1pdm09) or A/H1N1/PuertoRico/08/1934 without blocking M2 S31N. The compounds and their cocktails while not to be more toxic than amantadine might be useful for re-purposing of amantadine class of drugs in the case (i) of the prevalence of M2 L26F and or M2 V27A strains (ii) of an antigenic shift of the virus to M2 WT and (iii) because they inhibited a broad panel of M2 WT and M2 S31N viruses including the H1N1pdm09). (d) We showed that the mechanism of antiviral activity against A/California/07/2009 or A/PR/08/1934 and possibly also M2 WT viruses compared to WSN/33 viruses is not due to inhibition of an early stage of virus infection or a late stage of M2 channel function during endocytosis or inhibition of HA binding to host cells or a different pH for HA fusion or a lysosomotropic effect.

Mechanism of Proton Pumping in Complex I of the Mitochondrial Respiratory Chain

We propose a physical mechanism of conformation-induced proton pumping in mitochondrial Complex I. The structural conformations of this protein are modeled as the motion of a piston having positive charges on both sides. A negatively charged electron attracts the piston, moving the other end away from the proton site, thereby reducing its energy and allowing a proton to populate the site. When the electron escapes, elastic forces assist the return of the piston, increasing proton site energy and facilitating proton transfer. We derive the Heisenberg equations of motion for electron and proton operators and rewrite them in the form of rate equations coupled to the phenomenological Langevin equation describing piston dynamics. This set of coupled equations is solved numerically. We show that proton pumping can be achieved within this model for a reasonable set of parameters. The dependencies of proton current on geometry, temperature, and other parameters are examined.

Interaction of Liquid Lead Bismuth and Materials in Spallation Target

Material choices for liquid lead bismuth spallation target are some of austenitic stainless steel, ferrite martensitic steel and cold-worked austenitic stainless steel. In order to ensure materials resistance to irradiation and corrosion as well as compatibility with lead bismuth, it is appropriate to lower the incident proton current density and the process temperature, in which temperature range engineering design can control to work, especially in ADS (Accelerator-Driven nuclear transmutation System) concept. The lower limit temperature is determined from the physical melting temperature and the engineering efficiency of the steam generator involved in process control. The material related issues for liquid lead bismuth are mass loss by impinging secondary flow, wettability at the device interface for ultrasonic waves application, detachable control of the slag in the flowing system, stabilized electrical resistance between the material and the liquid lead bismuth interface. Electromagnetic fluid analyses show how flow rate relates electrical resistivity of flow channel material.

Novel Proton-Conducting Oxygen-Deficient Complex Oxides: Synthesis, Hydration Processes, Transport Properties

The complex oxide BaLaIn0.9Nb0.1O4.1 with Ruddlesden-Popper structure was obtained for the first time. It was found that the introduction of niobium into indium sublattice leads to the increase in the cell volume. Hydration processes and electrical properties have been investigated. For BaLaIn0.9Nb0.1O4.1 it was proved the capability for water uptake and the appearance of proton current carriers. It was established that niobium doping leads to the increase of conductivity compared to undoped composition BaLaInO4 at ~1 order of magnitude in whole temperature range.

Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

Real-time functional analysis of Hv1 channel in neutrophils: a new approach from zebrafish model

Voltage-gated proton channel (Hv1) has been studied in various immune cells, including neutrophils. However, most studies have taken an in vitro approach using isolated cells or primary cultured cells of mammals; therefore, limited evidence is available on the function of Hv1 in a physiological context. In this study, we have developed the in vivo system that enables real-time functional analysis of Hv1 using zebrafish embryos ( Danio rerio). Hvcn1-deficiency ( hvcn1−/−) in zebrafish completely abolished voltage-gated proton current, which is typically observed in wild-type neutrophils. Importantly, hvcn1-deficiency significantly reduced reactive oxygen species production and calcium response of zebrafish neutrophils, comparable to the results observed in mammalian models. These findings verify zebrafish Hv1 (DrHv1) as the primary contributor for native Hv1-derived proton current in neutrophils and suggest the conserved function of Hv1 in the immune cells across vertebrate animals. Taking advantage of Hv1 zebrafish model, we compared real-time behaviors of neutrophils between wild-type and hvcn1−/− zebrafish in response to tissue injury and acute bacterial infection. Notably, we observed a significant increase in the number of phagosomes in hvcn1−/− neutrophils, raising a possible link between Hv1 and phagosomal maturation. Furthermore, survival analysis of zebrafish larvae potentially supports a protective role of Hv1 in the innate immune response against systemic bacterial infection. This study represents the influence of Hv1 on neutrophil behaviors and highlights the benefits of in vivo approach toward the understanding of Hv1 in a physiological context.

Electrically-Induced Light Emission in Polymer-Stabilized Proton Gradient Systems

<div>Experiments with the chemically generated proton gradient, have lead to fabricate the system which emits the light, when electrically powered, owing to proton current flow. A polymeric scaffold preserves the stability of the system and, at the same time is playing a role of dopants of water, due to chemical character (acidic or basic) of side groups, which are responsible for gradient formation. The</div><div>results of experiments with the chemical model of the proton gradient, the protonic p-n junction, as a light emitting diode, make a “proof of concept”, which opens the way into new light generating process and new device (protonic LED), but also, to general understanding the proton gradient role in</div><div>the light emission processes.</div>

Electrically-Induced Light Emission in Polymer-Stabilized Proton Gradient Systems

<div>Experiments with the chemically generated proton gradient, have lead to fabricate the system which emits the light, when electrically powered, owing to proton current flow. A polymeric scaffold preserves the stability of the system and, at the same time is playing a role of dopants of water, due to chemical character (acidic or basic) of side groups, which are responsible for gradient formation. The</div><div>results of experiments with the chemical model of the proton gradient, the protonic p-n junction, as a light emitting diode, make a “proof of concept”, which opens the way into new light generating process and new device (protonic LED), but also, to general understanding the proton gradient role in</div><div>the light emission processes.</div>