Here, we report the first systematic study of two distinct configurations of hexagonal silver nanoparticle (AgNP) microdomain arrays prepared on polyethylene terephthalate (PET) substrates using the laser-induced forward transfer (LIFT) technique. By varying the size and spacing of the domains, we investigated how geometric parameters govern nanoparticle distribution, surface morphology, and biological response. AFM, SEM, CLSM, EDX, and XPS analyses revealed a pronounced gradient of AgNP density across the patterned surfaces, with the highest roughness and nanoparticle aggregation in domain centers, reduced coverage at the edges, and detectable but sparse particles in inter-domain gaps. Cross-section TEM further confirmed that nanoparticles are confined to a thin surface layer, ensuring high biological availability while minimizing uncontrolled leaching. Antibacterial activity assessment revealed that both Staphylococcus epidermidis (Gram-positive) and Escherichia coli (Gram-negative) were reduced below the detection limit within two hours across all patterned surfaces, irrespective of domain size. Cytotoxicity tests using normal human dermal fibroblasts (NHDF) revealed that the cells adhered, proliferated, and remained metabolically active over a seven-day period, although their enzymatic activity and numbers were reduced compared to cells growing on pristine PET. These findings highlight the unique dual functionality of LIFT-fabricated AgNP microdomains, which exhibit rapid and broad-spectrum antibacterial activity, combined with good cytocompatibility. The demonstrated ability to tune surface architecture by controlling domain size and spacing offers a promising pathway toward advanced antimicrobial coatings for medical devices and implants.

The development of advanced methods of SERS-CNN data analysis seems to provide a perfect analytical system that is capable of solving the sophisticated task of determining the species and the behavior of microorganisms. Unlike the widely-used analytical approach, machine learning allows precise analysis even of very complex spectra of biological samples, and can provide precise decisions for a specific biochemical or microbiological task. In this article, we show for the first time the utilization of the SERS–CNN approach for remote observation of mesenchymal stem cell behavior. Our approach is based on SERS measurements of the biochemical changes taking place in the surrounding culture media due to stem cell proliferation and their biochemical activity. The cells were cultivated on various substrates supporting random or oriented cell growth, and also on “surface-toxic” substrates. SERS-CNN analysis reveals the ability to perform “remote” non-invasive estimation (i.e. using the surrounding medium analysis) of the degree of cell survival and the proliferation rate, using Raman measurements and advanced spectra data processing. It should be noted that the proposed approach makes it possible to analyze cell behavior without disrupting cell growth, and it can also be performed by untrained staff with the use of widely-available equipment.

Damaged neural tissue is regenerated by neural stem cells (NSCs), which represent a rare and difficult-to-culture cell population. Therefore, alternative sources of stem cells are being tested to replace a shortage of NSCs. Here we show that mouse adipose tissue-derived mesenchymal stem cells (MSCs) can be effectively differentiated into cells expressing neuronal cell markers. The differentiation protocol, simulating the inflammatory site of neural injury, involved brain tissue extract, fibroblast growth factor, epidermal growth factor, supernatant from activated splenocytes and electrical stimulation under physiological conditions. MSCs differentiated using this protocol displayed neuronal cell morphology and expressed genes for neuronal cell markers, such as neurofilament light (Nf-L), medium (Nf-M) and heavy (Nf-H) polypeptides, synaptophysin (SYP), neural cell adhesion molecule (NCAM), glutamic acid decarboxylase (GAD), neuron-specific nuclear protein (NeuN), βIII-tubulin (Tubb3) and microtubule-associated protein 2 (Mtap2), which are absent (Nf-L, Nf-H, SYP, GAD, NeuN and Mtap2) or only slightly expressed (NCAM, Tubb3 and Nf-M) in undifferentiated cells. The differentiation was further enhanced when the cells were cultured on nanofibre scaffolds. The neural differentiation of MSCs, which was detected at the level of gene expression, was confirmed by positive immunostaining for Nf-L protein. The results thus show that the simulation of conditions in an injured neural tissue and inflammatory environment, supplemented with electrical stimulation under physiological conditions and cultivation of cells on a three-dimensional (3D) nanofibre scaffold, form an effective protocol for the differentiation of MSCs into cells with neuronal markers. Copyright © 2015 John Wiley & Sons, Ltd.

The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2′,5′-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

J. Neurochem. (2011) 119, 40–49.AbstractWe have studied the role of different amino acids in the M2 transmembrane domain of the α7 neuronal nicotinic receptor by mutating residues that differ from the ones located at the same positions in other α (α2–α10) or β (β2–β4) subunits. Our aim was to investigate the contribution of these amino acids to the peculiar kinetic and inward rectification properties that differentiate the homomeric α7 receptor from other nicotinic receptors. Mutations of several residues strongly modified receptor function. We found that Thr245 had the most profound effect when mutated to serine, an amino acid present in all heteromeric receptors composed of α and β subunits, by dramatically increasing the maximal current, decreasing the decaying rate of the currents and decreasing receptor rectification. Some mutants also showed altered agonist‐binding properties as revealed by shifts in the dose–response curves for acetylcholine. We conclude that residues in the M2 segment and flanking regions contribute to the unusual properties of the α7 receptor, especially to its characteristic fast kinetic behavior and strong inward rectification and furthermore to the potency of agonists.

The role of negatively charged amino acids in the F-loop of the beta 4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the alpha 3 beta 4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at -40 mV produced inward currents which displayed biphasic desensitization. The EC50 of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.

Physostigmine (Phy), a reversible inhibitor of acetylcholine (ACh) esterase (AChE), may also act as a low potency agonist and a modulator of the nicotinic receptor. The actions of Phy on mouse muscle nicotinic receptors in the COS-7 cell line were studied by the patch-clamp technique. Currents were recorded in the whole-cell mode 3-7 days after cell transfection by plasmids coding alphabetagammadelta combination of receptor subunits. The application of ACh to cells clamped at -10 mV produced inward currents which displayed desensitization. The application of Phy in concentrations up to 1 x 10(-3) M did not give reliable specific whole-cell membrane responses. The application of Phy in concentrations of 10(-6)-10(-4) M together with ACh modulated the amplitude; accelerated desensitization of currents induced by ACh and increased the final extent of desensitization in a concentration-dependent manner. This finding is in contrast to the suppression and slowing down of desensitization by Phy and 1-methyl-galanthamine observed in Torpedo receptors.

The action of physostigmine on mouse muscle nicotinic acetylcholine receptor expressed in the COS-7 cell line was studied by the patch-clamp technique. Physostigmine accelerated, by allosteric modulation, the rate of desensitization of whole cell currents induced by acetylcholine and decreased the maximal amplitude in concentration-dependent manner.

A bstract : The action of physostigmine on mouse muscle nicotinic acetylcholine receptor expressed in the COS-7 cell line was studied by the patch-clamp technique. Physostigmine accelerated, by allosteric modulation, the rate of desensitization of whole cell currents induced by acetylcholine and decreased the maximal amplitude in concentration-dependent manner.

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.