Tunable Self-Assembled Peptide Hydrogel Sensor for Pharma Cold Supply Chain.

Defrost sensors are a crucial element for proper functioning of the pharmaceutical cold chain. In this paper, the self-assembled peptide-based hydrogels were used to construct a sensitive defrost sensor for the transportation and storage of medications and biomaterials. The turbidity of the peptide hydrogel was employed as a marker of the temperature regime. The gelation kinetics under different conditions was studied to detect various stages of hydrogel structural transitions aimed at tuning the system properties. The developed sensor can be stored at room temperature for a long period, irreversibly indicates whether the product has been thawed, and can be adjusted to a specific temperature range and detection time.

Sensing Cells-Peptide Hydrogel Interaction In Situ via Scanning Ion Conductance Microscopy.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.

Mechanical Enhancement and Kinetics Regulation of Fmoc-Diphenylalanine Hydrogels by Thioflavin†T.

The self-assembly of peptides is a key direction for fabrication of advanced materials. Novel approaches for fine tuning of macroscopic and microscopic properties of peptide self-assemblies are of a high demand for constructing biomaterials with desired properties. In this work, while studying the kinetics of the Fmoc-Diphenylalanine (Fmoc-FF) dipeptide self-assembly using the Thioflavin T (ThT) dye, we observed that the presence of ThT strongly modifies structural and mechanical properties of the Fmoc-FF hydrogel. Notably, the presence of ThT resulted in a tenfold increase of the gelation time and in the formation of short and dense fibers in the hydrogel. As a result of these morphological alteration higher thermal stability, and most important, tenfold increase of the hydrogel rigidity was achieved. Hence, ThT not only slowed the kinetics of the Fmoc-FF hydrogel formation, but also strongly enhanced its mechanical properties. In this study, we provide a detailed description of the ThT effect on the hydrogel properties and suggest the mechanisms for this phenomenon, paving the way for the novel approach to the control of the peptide hydrogels' micro- and macroscale properties.

Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

Modulation of Proton-Gated Channels by Antidepressants.

The chemical structures of some antidepressants are similar to those of recently described amine-containing ligands of acid-sensing ion channels (ASICs). ASICs are expressed in brain neurons and participate in numerous CNS functions. As such, they can be related to antidepressant action or side effects. We therefore studied the actions of a series of antidepressants on recombinant ASIC1a and ASIC2a and on native ASICs in rat brain neurons. Most of the tested compounds prevented steady-state ASIC1a desensitization evoked by conditioning acidification to pH 7.1. Amitriptyline also potentiated ASIC1a responses evoked by pH drops from 7.4 to 6.5. We conclude that amitriptyline has a twofold effect: it shifts activation to less acidic values while also shifting steady-state desensitization to more acidic values. Chlorpromazine, desipramine, amitriptyline, fluoxetine, and atomoxetine potentiated ASIC2a response. Tianeptine caused strong inhibition of ASIC2a. Both potentiation and inhibition of ASIC2a were accompanied by the slowdown of desensitization, suggesting distinct mechanisms of action on activation and desensitization. In experiments on native heteromeric ASICs, tianeptine and amitriptyline demonstrated the same modes of action as on ASIC2a although with reduced potency.

Dissection of the deep-blue autofluorescence changes accompanying amyloid fibrillation.

Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (∼350 nm excitation; ∼450 nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.

Potentiation and Block of ASIC1a by Memantine.

Acid-sensing ion channels (ASICs) are modulated by various classes of ligands, including the recently described hydrophobic monoamines, which inhibit and potentiate ASICs in a subunit-specific manner. In particular, memantine inhibits ASIC1a and potentiates ASIC2a homomers. The aim of the present work was to characterize action mechanism of memantine on recombinant ASIC1a expressed in CHO (Chinese hamster ovary) cells. We have demonstrated that effect of memantine on ASIC1a strongly depends on membrane voltage, conditioning pH value and application protocol. When applied simultaneously with activating acidification at hyperpolarized voltages, memantine caused the strongest inhibition. Surprisingly, application of memantine between ASIC1a activations at zero voltage caused significant potentiation. Analysis of the data suggests that memantine produces two separate effects, voltage-dependent open-channel block and shift of steady-state desensitization curve to more acidic values. Putative binding sites are discussed based on the computer docking of memantine to the acidic pocket and the pore region.

Binding of thioflavin T by albumins: An underestimated role of protein oligomeric heterogeneity.

Amyloid fibrils formation is the well-known hallmark of various neurodegenerative diseases. Thioflavin T (ThT)-based fluorescence assays are widely used to detect and characterize fibrils, however, if performed in bioliquids, the analysis can be biased due to the presence of other, especially abundant, proteins. Particularly, it is known that albumin may bind ThT, although the binding mechanism remains debatable. Here the role of low-order albumin oligomers in ThT binding is investigated using time-resolved fluorometry and size-exclusion chromatography. Under conditions used, the fraction of dimers in human (HSA) and bovine (BSA) serum albumin solutions is as low as ∼7%, however, it is responsible for ∼50% of ThT binding. For both albumins, the binding affinity was estimated to be ∼200 and ∼40µM for monomeric and dimeric species, respectively. Molecular docking suggested that ThT preferentially binds in the hydrophobic pocket of subdomain IB of albumin monomer in a similar position but with a variable torsion angle, resulting in a lower fluorescence enhancement (∼40-fold) compared to amyloid fibrils (∼1000-fold). Dimerization of albumin presumably creates an extra binding site at the subunit interface. These results demonstrate the underestimated role of low-order albumin oligomers that can be highly relevant when analyzing drugs binding using fluorescence spectroscopy.

Cell Type- and Layer-Specific Muscarinic Potentiation of Excitatory Synaptic Drive onto Parvalbumin Neurons in Mouse Prefrontal Cortex.

Cholinergic neuromodulation is thought to shape network activity in the PFC, and thus PFC-dependent cognitive functions. ACh may modulate the activity of parvalbumin-positive (PV+) neurons, which critically regulate cortical network function. However, the mechanisms of cholinergic regulation of PV+ neuron activity, and particularly of the basket cell (BC) versus chandelier cell (ChC) subtypes, are unclear. Using patch clamp recordings in acute slices, we examined the effects of the ACh receptor (AChR) agonist carbachol on the excitatory synaptic drive onto BCs or ChCs in layers 2 to 6 of mouse PFC. Carbachol increased the frequency and amplitude of spontaneous EPSCs (sEPSCs) recorded from PV+ BCs in layers 3-6, but not in BCs from layer 2. Moreover, carbachol did not change the sEPSCs in ChCs, which were located exclusively in layer 2. The potentiation of sEPSCs in layers 3-6 BCs was prevented by the Na+ channel blocker tetrodotoxin and was abolished by the M1-selective muscarinic AChR antagonist pirenzepine. Thus, carbachol potentiates the activity-dependent excitatory drive onto PV+ neurons via M1-muscarinic AChR activation in a cell type- and layer-specific manner. In current clamp recordings with synaptic transmission blocked, carbachol directly evoked firing in deep layer pyramidal neurons (PNs). In contrast, carbachol elicited deep layer BC firing indirectly, via glutamate-mediated synaptic drive. Our data suggest that ACh powerfully regulates PFC microcircuit function by facilitating the firing of PNs that synaptically recruit deep layer PV+ BC activity, possibly shaping the patterns of network activity that contribute to cognitive function.

Effect of social interactions on hippocampal protein expression in animal dominant and submissive model of behavioral disorders.

PURPOSE: Psychiatric conditions, in many cases, arise from social interactions necessary for optimal mental functioning. Dominance and submissiveness are two opposite poles of behavior, stemming from processes of social interactions between members inside one group or species. Extreme dominance and submissiveness expressions in humans is accompanied by mental impairments, including mania and depression. Here, taking advantage of animals bred selectively for traits of dominance and submissiveness, we assess protein expression profiles in dominant and submissive mice in the context of social interaction. EXPERIMENTAL DESIGN: Proteins extracted from hippocampi of naïve and social interaction subjected dominant, submissive and wild type mice (15 mice per each group) are quantified using label-free quantitative LC/MS/MS analysis. Complexity of social interaction-related protein expression is resolved by factor analysis and enriched with GO and protein-protein interaction functional network analyses. RESULTS: In total, 1146 proteins exhibiting expression changes in the wild type mice, as well as dominant and submissive mice are enriched in protein datasets responsible for: 1) socially triggered dominance (90 proteins), 2) inherent submissiveness (75 proteins), 3) socially triggered submissiveness (117 proteins), and 4) social interaction triggered protein expression changes, related to resilience/adaptation to stress (69 proteins). Among the most enriched categories, extensive changes are found in proteins related to presynaptic release, ion channel regulation, circadian rhythm, MAPK, ErbB and NF-kB pathways. CONCLUSION: Data extracted from this first extensive proteomic study of a social interaction paradigm may facilitate decoding of molecular mechanisms responsible for pathogenesis of psychiatric disorders.

Thermally induced conformational changes and protein-protein interactions of bovine serum albumin in aqueous solution under different pH and ionic strengths as revealed by SAXS measurements.

Thermal-induced conformational changes and protein-protein interactions of bovine serum albumin (BSA) in aqueous solution are assessed by small angle X-ray scattering (SAXS) at two pH values (7.4 and 9.0) and two ionic strengths (0.1 and 0.5). We demonstrate that Guinier analysis in two ranges of the modulus of the scattering vector allows protein melting and aggregation to be monitored simultaneously, thus providing insights into the mechanism of thermal-induced BSA aggregation. Results of the analysis suggest that at room temperature monomeric and dimeric BSA fractions are present in solution. For low concentrations (<10 mg mL-1) the monomeric to dimeric fraction ratio is close to 6, the same value we obtained independently in size-exclusion chromatography experiments. For elevated concentrations (20 mg mL-1 and 40 mg mL-1) a decrease in the dimer fraction occurs. Following heating, dimer formation is observed prior to protein melting, while no higher order aggregates are observed in the 20-60 °C temperature range. In the vicinity of the BSA melting point, higher order aggregates appear and protein molecules exhibit an aggregation burst. Higher ionic strength makes the described effects more pronounced - dimer formation increases at lower temperatures, presumably due to partial screening of electrostatic interactions between protein molecules. Moreover, the melting temperature shifts to higher values upon increasing the protein concentration and pH, indicating that repulsive interactions stabilize the protein structure. The suggested model was verified by the assessment of parameters of protein-protein interaction potentials based on DLVO theory using the global fitting procedure.

Histamine selectively potentiates acid-sensing ion channel 1a.

Although acid-sensitive ion channels (ASICs) play an important role in brain functions, the exact mechanism of their physiological activation remain unclear. A possible answer to the intriguing question is that some presently unknown endogenous ligand(s) positively modulate ASICs and enhance their responses to physiologically significant level. In the present work we found that histamine selectively potentiates ASIC1a homomers in CHO cells. Action of histamine was particularly pronounced at modest acidifications, which cause minor response. At these conditions micromolar concentrations of histamine have provided significant potentiation of ASIC1a response. We proposed that histamine and possibly some other endogenous amines can positively modulate ASICs functions.

The role of colloid particles in the albumin-lanthanides interaction: The study of aggregation mechanisms.

We studied the interaction between bovine serum albumin (BSA) and lanthanide ions in aqueous solution in the 4.0÷9.5pH range. A strong increase of the solution turbidity was observed at pH values exceeding 6, which corresponds to the formation of Ln(OH)3 nanoparticles, while no changes were observed near the isoelectric point of BSA (pH 4.7). The results of the dynamic light scattering and protein adsorption measurements clearly demonstrated that the observed turbidity enhancement was caused by albumin sorption on the surface of Ln(OH)3 and colloid particles bridging via adsorbed protein molecules. Upon pH increase from 4.5 to 6.5, albumin adsorption on lanthanide colloids was observed, while the following increase of pH from 6.5 to 9.5 led to protein desorption. The predominant role of the electrostatic interactions in the adsorption and desorption processes were revealed in the zeta-potential measurements. No reversibility was observed upon decreasing pH from 9.5 to 4.5 that was suggested to be due to the other interaction mechanisms present in the system. It was shown that while for all lanthanide ions the interaction mechanism with BSA was similar, its manifestation in the optical properties of the system was significantly different. This was interpreted as a consequence of the differences in lanthanides hydrolysis constants.

Knowledge Management within the Medical University.

The aim of the work is studying the possibilities of ontological engineering in managing of medical knowledge. And also practical implementation of knowledge management system (KMS) in medical university. The educational process model is established that allows analyzing learning results within time scale. Glossary sub-system has been developed; ontologies of educational disciplines are constructed; environment for setup and solution of situational cases is established; ontological approach to assess competencies is developed. The possibilities of the system for solving situation tasks have been described. The approach to the evaluation of competence has been developed.

Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways.

We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

Non-classical mechanism of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor channel block by fluoxetine.

Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca(2+)-permeable and Ca(2+)-impermeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca(2+)-impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC50 value for the inhibition of Ca(2+)-permeable AMPA receptors in giant striatal interneurons was 43 ± 7 µM. The inhibition of Ca(2+)-permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open-channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane-impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open-channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.

Monoamine NMDA receptor channel blockers inhibit and potentiate native and recombinant proton-gated ion channels.

Acid-sensing ion channels (ASICs) are widely distributed in the peripheral and central nervous system. Although they are involved in many physiological functions, the actual processes that activate ASICs remain unclear. This is particularly true for brain ASICs, which produce only a transient response to a fast drop in pH and cannot mediate sustained current. Therefore, the search for ASIC inhibitors and, especially, potentiators/activators is important. We report that NMDA receptor channel blockers with a comparatively simple structure (9-aminoacridine, memantine, IEM-2117 and IEM-1921) potentiate and/or inhibit ASICs in submillimolar concentrations. The experiments were performed using the patch clamp technique on native ASICs from rat hippocampal interneurons and recombinant ASICs of different subunit compositions expressed in CHO cells. Native ASICs were potentiated by IEM-1921 and IEM-2117, and inhibited by memantine and 9-aminoacridine. Homomeric ASIC1a were inhibited by memantine, IEM-2117 and 9-aminoacridine while IEM-1921 was ineffective. In contrast, homomeric ASIC2a were potentiated by IEM-2117, memantine and IEM-1921, whereas 9-aminoacridine was inactive. The compounds caused a complex effect on ASIC3. 9-aminoacridine and IEM-1921 potentiated the steady-state response of ASIC3 and inhibited the peak component. IEM-2117 not only potentiated ASIC3-mediated currents caused by acidification but also evoked steady-state currents at neutral pH. Our results demonstrate that, depending on the subunit composition, ASICs can be activated or inhibited by simple compounds that possess only amino group and aromatic/hydrophobic moieties. This opens up the possibility to search for new ASIC modulators among a number of endogenous ligands.

Assessment of the europium(III) binding sites on albumin using fluorescence spectroscopy.

Intrinsic fluorescence quenching of bovine serum albumin (BSA) and europium(III) luminescence in BSA complexes were investigated. The number of BSA binding sites (n) and equilibrium constant (Keq) values were determined from both measurements provided qualitatively different results. While the modified Stern-Volmer relation for BSA fluorescence quenching gave n = 1 at pH 4.5 and pH 6, two sets of binding sites were determined from Eu(3+) luminescence with n1 = 2, n2 = 4 at pH 6 and n1 = 1, n2 = 2 at pH 4.5. The model explaining the discrepancy between the results obtained by these fluorescent approaches was suggested, and the limitations in application of the "log-log" Stern-Volmer plots in analysis of binding processes were discussed.