Olfactory dysfunction in SARS-CoV-2 infection: Focus on odorant specificity and chronic persistence.

BACKGROUND: Smell dysfunction has been recognized as an early symptom of SARS-CoV-2 infection, often occurring before the onset of core symptoms of the respiratory tract, fever or muscle pain. In most cases, olfactory dysfunction is accompanied by reduced sense of taste, is partial (microsmia) and seems to normalize after several weeks, however, especially in cases of virus-induced complete smell loss (anosmia), there are indications of persisting deficits even 2 months after recovery from the acute disease, pointing towards the possibility of chronic or even permanent smell reduction for a significant part of the patient population. To date, we have no knowledge on the specificity of anosmia towards specific odorants or chemicals and about the longer-term timeline of its persistence or reversal. METHODS: In this longitudinal study, 70 participants from a community in Lower Austria that had been tested positive for either IgG or IgM SARS-CoV-2 titers in June 2020 and a healthy control cohort (N = 348) underwent smell testing with a 12-item Cross-Cultural Smell Identification Test (CC-SIT), based upon items from the University of Pennsylvania Smell Identification Test (UPSIT). The test was performed in October 2020, i.e. 4 months after initial diagnosis via antibody testing. Results were analyzed using statistical tests for contingency for each smell individually in order to detect whether reacquisition of smell is dependent on specific odorant types. RESULTS: For all odorants tested, except the odor "smoke", even 4 months or more after acute SARS-CoV-2 infection, participants with a positive antibody titer had a reduced sense of smell when compared to the control group. On average, while the control cohort detected a set of 12 different smells with 88.0% accuracy, the antibody-positive group detected 80.0% of tested odorants. A reduction of accuracy of detection by 9.1% in the antibody-positive cohort was detected. Recovery of the ability to smell was particularly delayed for three odorants: strawberry (encoded by the aldehyde ethylmethylphenylglycidate), lemon (encoded by citronellal, a monoterpenoid aldehyde), and soap (alkali metal salts of the fatty acids plus odorous additives) exhibit a sensitivity of detection of an infection with SARS-CoV-2 of 31.0%, 41.0% and 40.0%, respectively. CONCLUSION: Four months or more after acute infection, smell performance of SARS-CoV-2 positive patients with mild or no symptoms is not fully recovered, whereby the ability to detect certain odors (strawberry, lemon and soap) is particularly affected, suggesting the possibility that these sensitivity to these smells may not only be lagging behind but may be more permanently affected.

A surface plasmon field-enhanced fluorescence reversible split aptamer biosensor.

Surface plasmon field-enhanced fluorescence is reported for the readout of a heterogeneous assay that utilizes low affinity split aptamer ligands. Weak affinity ligands that reversibly interact with target analytes hold potential for facile implementation in continuous monitoring biosensor systems. This functionality is not possible without the regeneration of more commonly used assays relying on high affinity ligands and end-point measurement. In fluorescence-based sensors, the use of low affinity ligands allows avoiding this step but it imposes a challenge associated with the weak optical response to the specific capture of the target analyte which is also often masked by a strong background. The coupling of fluorophore labels with a confined field of surface plasmons is reported for strong amplification of the fluorescence signal emitted from the sensor surface and its efficient discrimination from the background. This optical scheme is demonstrated for time-resolved analysis of chosen model analytes - adenoside and adenosine triphosphate - with a split aptamer that exhibits an equilibrium affinity binding constant between 0.73 and 1.35 mM. The developed biosensor enables rapid and specific discrimination of target analyte concentration changes from low µM to mM in buffer as well as in 10% serum.

Influence of myelin proteins on the structure and dynamics of a model membrane with emphasis on the low temperature regime.

Myelin is an insulating, multi-lamellar membrane structure wrapped around selected nerve axons. Increasing the speed of nerve impulses, it is crucial for the proper functioning of the vertebrate nervous system. Human neurodegenerative diseases, such as multiple sclerosis, are linked to damage to the myelin sheath through demyelination. Myelin exhibits a well defined subset of myelin-specific proteins, whose influence on membrane dynamics, i.e., myelin flexibility and stability, has not yet been explored in detail. In a first paper [W. Knoll, J. Peters, P. Kursula, Y. Gerelli, J. Ollivier, B. Demé, M. Telling, E. Kemner, and F. Natali, Soft Matter 10, 519 (2014)] we were able to spotlight, through neutron scattering experiments, the role of peripheral nervous system myelin proteins on membrane stability at room temperature. In particular, the myelin basic protein and peripheral myelin protein 2 were found to synergistically influence the membrane structure while keeping almost unchanged the membrane mobility. Further insight is provided by this work, in which we particularly address the investigation of the membrane flexibility in the low temperature regime. We evidence a different behavior suggesting that the proton dynamics is reduced by the addition of the myelin basic protein accompanied by negligible membrane structural changes. Moreover, we address the importance of correct sample preparation and characterization for the success of the experiment and for the reliability of the obtained results.

Long-range attraction and molecular rearrangements in receptor-ligand interactions.

A surface force apparatus was used to measure a long-range attractive protein-ligand force at separations D less than 85 angstroms. This force may effectively "steer" ligand trajectories, resulting in a greater than 27-fold enhancement of the association rate. A much stronger specific attraction is measured at contact (D less than 4 angstroms). A sevenfold increase in intermembrane adhesion resulted from increased lateral mobility of the receptors and molecular rearrangements in membranes above the solid-fluid transition temperature.

Attempts to mimic docking processes of the immune system: recognition-induced formation of protein multilayers.

The assemblage of protein multilayers induced by molecular recognition, as seen, for example, in the immune cascade, has been mimicked by using streptavidin as a docking matrix. For these experiments, this protein matrix was organized on liposomes, monolayers at the air-water interface, and self-assembled layers on gold, all three containing biotin lipids. The docking of streptavidin to biotin at liposomal surfaces was confirmed by circular dichroism. Mixed double and triple layers of streptavidin, concanavalin A, antibody Fab fragments, and hormones are prepared at the air-water interface and on gold surfaces and were characterized by fluorescence microscopy and plasmon spectroscopy. With the use of biotin analogs that have lower binding constants it has been possible to achieve multiple formation and competitive replacement of the oriented protein assemblages.

Anisotropic propagation and confinement of high frequency phonons in nanocomposites.

We show that self-ordered anodic aluminum oxide containing hexagonal arrays of cylindrical nanopores with submicron periodicity is a versatile model system for the exploration of rich phononic phenomena at gigahertz frequencies, which are intimately linked to fluids located in the nanopores and their interactions with the pore walls. Using high-resolution Brillouin spectroscopy we report the first realization of directional flow of elastic energy parallel and perpendicular to the pore axes, phonon localization, and tunability of the phononic band structure.

Preparation of gold nanoparticles in an aqueous medium using 2-mercaptosuccinic acid as both reduction and capping agent.

Monolayer protected gold nanoparticles with diameters above 10 nm were prepared by a simple, one step reaction in water. 2-mercaptosuccinic acid (MSA) was used both as reduction agent for hydrogen tetrachloroaurate (HAuCl4) and as stabilizing agent for the gold nanoparticles. Size distribution and surface chemistry were investigated by UV-Vis spectroscopy, scanning electron microscopy and Fourier Transform Infrared Spectroscopy. Particle size can be controlled by adjusting the molar portions of the reactants. The resulting particles are efficiently stabilized against aggregation when MSA is used in a concentration of 40% and above. Below a minimum MSA concentration a long-term particle growth is observed.

Raman-spectroscopic evidence for the incorporation of alamethicin into dimyristoylphosphatidylcholine bilayers.

The interaction of alamethicin with dimyristoylphosphatidylcholine dispersions was investigated by Raman spectroscopy. The temperature dependence of the C-H stretching spectra demonstrates the incorporation of the polypeptide into the hydrophobic core even in the absence of a transmembrane potential gradient.

pH-control of the miscibility properties of a binary lipid alloy and its influence on the ion transport by gramicidin.

We studied the coupling of a membrane function (the transport of ions by the pore forming polypeptide gramicidin) to chemically driven phase changes in black membranes of binary lipid mixtures. In particular, we investigated the influence of the aqueous pH value on the fluid-fluid demixing effect of Ca2+ to phosphatidylcholine/phosphatidylglycerol bilayers. It is found that one can switch, under certain conditions, between a homogeneously mixed and a phase separated membrane by changing the pH. We interpret this as being caused by the change in the degree of dissociation of one of the lipid components.

Ca(2+)-induced lateral phase separation in black lipid membranes and its coupling to the ion translocation by gramicidin.

We analyze the single-channel current fluctuations of gramicidin incorporated into biomolecular lipid membranes (BLM) of binary mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) as a function of the Ca2+ concentration in the electrolyte (0.5 M CsCl, pH 6) solution. At low Ca2+ levels (cCa2+ < 10(-6) M) a monomodal conductance histogram and a single average lifetime suggests a homogeneous mixture over the full range of composition (PG(1-x)PCx, 0 < or = x < or = 1). At higher Ca2+ concentrations phase separation processes are inferred from the appearance of bimodal conductance histograms. The two channel populations (in the two coexisting phases) can also be distinguished through their different average lifetimes. By a systematic variation of the mole fractions of the two lipid components we derive the respective phase boundaries and thus the full Ca2+ concentration-composition phase diagram.

Ca2+-induced phase separation in black lipid membranes and its effect on the transport of a hydrophobic ion.

Voltage jump-current relaxation studies have been performed with dipicrylamine-doped black membranes of binary lipid mixtures. As in the case of the carrier-mediated ion transport (Schmidt, G., Eibl, H. and Knoll, W. (1982) J. Membrane Biol. 70, 147-155) no evidence was found that the neutral lipid phosphatidylcholine (DPMPC) and the charged phosphatidic acid (DPMPA) are heterogeneously distributed in the membrane over the whole range of composition. However, besides a continuous dilution of the surface charges of DPMPA by the addition of DPMPC molecules, different structural properties of mixed membranes influence the kinetics of the dipicrylamine transport. The addition of Ca2+ to the electrolyte induces a lipid phase separation within the membrane into two fluid phases of distinctly different characteristics of the translocation of hydrophobic ions. Thus, it is possible to determine a preliminary composition phase diagram for the DPMPA/DPMPC mixtures as a function of the Ca2+ concentration.

Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram.

Small-angle neutron scattering has been used to study structural features of lamellar bilayer membranes of dimyristoylphosphatidylcholine (DMPC) and DMPC mixed with various amount of cholesterol. The studies were recorded at a fixed hydration level of 17% 2H2O, i.e. just below saturation. Bragg reflections gives information on the ripple structure and on the bilayer periodicity. The crystalline Lc phase, which was stabilized after long time storage at low temperature, exhibits major small angle scattering when cholesterol is mixed into the membrane. The intermediate P beta' gel-phase, which is characteristic by the rippled structure, is dramatically stabilized by the introduction of cholesterol. The ripple structure depends significantly both on the cholesterol content and on the temperature. At high temperatures, T greater than 15 degrees C, the inverse ripple periodicity varies basically linearly with cholesterol content, and approach zero (i.e. periodicity goes to infinite) at 20 mol% cholesterol, approximately. At lower temperatures the correlation is more complex. The data indicate additional phase boundaries below 2 mol% and at approx. 8 mol%. Secondary rippled structures are observed in the low temperature L beta'-phase for cholesterol content below approx. 8 mol%. The data gives detailed insight into the phosphatidylcholine cholesterol phase diagram, which is discussed on the basis of a simple model in which the cholesterol complexes are fixed to the defect stripes of the rippled structure.

Electron microscopic investigations on free-standing mixed lipid Langmuir-Blodgett-Kuhn monolayers: phase separation and aging process.

Lipid monolayers were prepared by the Langmuir-Blodgett-Kuhn technique (LBK) as free-standing films spanning a diameter of up to 1 micron. These films were investigated by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The free-standing monolayer is shown to be in a transient state in which an aging process is proceeding: after storage for two weeks in air at room temperature the films tear off the edges of the perforated supporting film. Ca2+ ions induced lateral phase separation in these films prepared from a 50:50 mixture of lecithin/glycerol could be visualized by means of ESI, i.e., by comparing micrographs below and above the Ca absorption edge in the EEL spectrum. The domain sizes of the demixed phases were determined to vary between 30 and 60 nm. In addition it was shown that the counter ion of the negatively charged glycerol in these films is Ca2+ and not Na+.

Translocation of alkali metal cations by lipophilic cyclodextrin derivatives through black lipid membranes.

Lipophilic cyclodextrin (CD) derivatives, synthetic ionophores, were prepared to transport alkali metal cations across a black lipid membrane (BLM). The purpose of this study is to develop a new class of an artificial transportation system of alkali metal cations via bilayer lipid membranes, by using CD derivatives as a cation carrier. A lipophilic CD derivative incorporated into a BLM forms a complex with an alkali metal cation at one surface of the membrane. This charged complex migrates to the opposite side of the membrane and then releases the cation into the subphase. CD derivatives have various types of acyl groups as a complexing site and formed a 1:1 complex with the alkali metal cation. The complex formation was interpreted by an induced-fit mechanism. It is found that the ability of CD derivative for forming a complex and/or transporting cations across the BLM depends on the bulkiness of acyl groups. The conductivities of heptakis (2,6-di-O-propyl-3-O-propionyl)-beta-CD were higher than those of valinomycin regardless of sizes of cations. The order of the conductivity in all derivatives is Li+ < Na+ < K+ approximately = Rb+ approximately = Cs+, regardless of the types of acyl groups in the derivatives. The effects of alkali metal cation concentration in the aqueous phase and CD concentration in the membrane on the translocation are also discussed.

Functional tethered membranes.

Phospholipid bilayer membranes at the interface between a substrate and an aqueous phase, supported by or tethered to the solid surface via a polymer cushion, a peptide-, protein-, or oligosaccharide-coupling layer have reached a stage at which they are important as a novel model membrane system but also offer potential for practical applications (e.g. for biosensing purposes with membrane-integral receptors). We briefly summarize some of the recent progress made in the structural characterization of the build-up of these rather complex interfacial architectures, in the functionalization of the pure lipid matrix by the reconstitution of proteins, and in the lateral patterning of the membranes as a prerequisite for the construction of membrane chips for massive parallel monitoring of binding events.

Independence of synaptic specificity from neuritic guidance.

Neuronal circuits are interconnected with a high degree of specificity. While axonal guidance has been demonstrated to be crucial for the choice of the correct target region, its role in specificity at the level of individual cells remains unclear. Specificity of synapse formation may either result from precise guidance of axonal outgrowth onto the target or depend on a molecular "match" between pre- and postsynapse. To distinguish between these possibilities, an in vitro system was used in which neuritic outgrowth of rat cortical neurons is accurately guided along the narrow pathways of a surface micropattern. The micropattern consisted of a blend of extracellular matrix molecules applied to a cell repellent background of polystyrene by microcontact printing. The system reproduces guidance by attractant and repellent surface cues while no other signals that may influence synapse formation, like gradients of trophic factors or accumulations of signaling molecules, are provided. While the number of contact points between neighboring cells was strongly reduced on patterned substrates due to the geometrical restrictions, frequency of synapse formation was not different from homogeneous cultures. Thus it was unaffected by stringent guidance onto the target cell or by the number of cell-cell contacts. Moreover, a statistically significant enrichment of reciprocal contacts between mixed pairs of excitatory and inhibitory neurons over probabilistic predictions was found, which has similarly been shown by others in dissociated neuronal cultures. Our results indicate that precise axonal guidance is insufficient for target-specific synapse formation and suggest that instead recognition between individual cells is required.

Cell-transistor hybrid systems and their potential applications.

Electrogenic cells fire spontaneous or triggered action potentials (transient changes of their membrane potentials) and can be electronically coupled to external electrodes (arrays). Signals from rat heart-muscle cells were recorded by a field-effect transistor and the results described on the basis of an equivalent circuit. This technique has potential applications in drug screening, such as measuring the dose-response curve of isoproterenol, a beta-adrenergic agonist with a positive chronotropic effect.

Lateral order in mixed lipid bilayers and its influence on ion translocation by gramicidin: a model for the structure-function relationship in membranes.

The temperature-composition phase diagram of dimyristoylphosphatidylcholine and dipentadecylphosphatidylglycerol (DiC15PG) was determined by mass densitometry. For a mixture containing 30 mol% DiC15PG, the homogeneous distribution of the 2 components is demonstrated in the fluid state at T = 35 degrees C by small-angle neutron scattering in combination with the inverse contrast variation method. By the same technique, the coexistence of fluid and condensed phases at T = 23.3 degrees C could be shown in agreement with the densitometric data. Furthermore, it is demonstrated that Ca++ induces, even at T = 35 degrees C, separation into 2 fluid phases. A corresponding phase separation is found in bimolecular lipid membranes ("black films") by analysis of the single-channel conductance fluctuations of gramicidin A incorporated into an equimolarly mixed membrane of neutral lecithin and charged phosphatidic acid. The results are discussed as primary examples on the model-membrane level for the important structure-function relationship of biomembranes.

Electrophysiological recordings of patterned rat brain stem slice neurons.

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information in the past. However, although the culture of brain slices has many advantages over dissociated neuronal cultures, its feasibility on patterned substrates has not been demonstrated to date. In the present study, neuronal outgrowth from brain stem slices onto homogeneous control substrates, and onto laminin structures of grid- and line-shape was achieved. Cultures were evaluated by means of phase contrast microscopy, antibody staining, and patch-clamp measurements. Only patterns with line sizes of more than 4 microm yielded satisfactory neuronal outgrowth. The size of the nodes in the pattern influenced the nodal compliance of the spreading cells and the amount of unstructured overgrowth. Best grid patterns were 4 microm lines and 10 microm nodes, best line patterns were 4 microm lines and 20 microm nodes. On patterned substrates, average sodium and potassium currents were reduced by approximately 50% compared to controls, whereas area-normalized ion-currents were in the same order of magnitude. This indicates that as a consequence of the pattern-enforced geometrical confinement, neurons tend to have a smaller surface. In addition, neurons on patterned substrates were rapidly covered with glial overgrowth. This was shown by antibody staining.

Host-guest interactions at self-assembled monolayers of cyclodextrins on gold

We have developed synthesis routes for the introduction of short and long dialkylsulfides onto the primary side of alpha-, beta-, and gamma-cyclodextrins. Monolayers of these cyclodextrin adsorbates were characterized by electrochemistry, wettability studies, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (TOF-SIMS), and atomic force microscopy (AFM). The differences in thickness and polarity of the outerface of the monolayers were measured by electro-chemistry and wettability studies. On average about 70% of the sulfide moieties were used for binding to the gold, as measured by XPS. Tof-SIMS measurements showed that the cyclodextrin adsorbates adsorb without any bond breakage. AFM measurements revealed for beta-cyclodextrin monolayers a quasi-hexagonal lattice with a lattice constant of 20.6 A, which matches the geometrical size of the adsorbate. The alpha-cyclodextrin and gamma-cyclodextrin monolayers are less ordered. Interactions of the anionic guests 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) and 2-(p-toluidinyl)naphthalene-6-sulfonic acid (2,6-TNS) and the highly ordered monolayers of heptapodant beta-cyclodextrin adsorbates were studied by surface plasmon resonance (SPR) and electrochemical impedance spectroscopy. The SPR measurements clearly showed interactions between a beta-cyclodextrin monolayer and 1,8-ANS. Electrochemical impedance spectroscopy measurements gave high responses even at low guest concentrations (< or = 5 microM). The association constant for the binding of 1,8-ANS (K = 289,000 +/- 13,000M-1) is considerably higher than the corresponding value in solution. (Partial) methylation of the secondary side of the beta-cyclodextrin strongly decreases the binding.