Antimicrobial peptides (AMPs) represent valid alternatives to conventional antibiotics primarily due to their mechanism of action, which consists of cytoplasmic membrane disruption. However, their clinical application is often limited by cytotoxicity at high concentrations and low intrinsic biostability. To address these limitations, various biochemical approaches have been explored. In recent years, the frog‐skin derived AMP Esc(1‐21) has been extensively characterized for its potent antimicrobial activity, especially against Gram‐negative bacteria, both in vitro and in vivo. In this study, we designed and synthesized novel Esc(1‐21) analogs in which a single isopeptide bond was introduced in place of a conventional peptide bond at specific positions within the sequence. The resulting five analogs were evaluated for their (i) chemical and structural properties, (ii) resistance to proteolytic degradation, (iii) antimicrobial and antibiofilm activities, (iv) hemolytic and cytotoxic effects, and (v) ability to perturb bacterial cytoplasmic membranes. Among these, Esc(1‐21)ε20 showed the most promising features, maintaining antimicrobial and antibiofilm activities comparable to those of the parent peptide while exhibiting lower cytotoxicity towards eukaryotic cells at higher concentrations and greater resistance to enzymatic degradation. These findings highlight Esc(1‐21)ε20 as an attractive lead candidate for the development of new antibiotic therapeutics.

The dielectric properties of biological cells provide critical insights into cellular physiology and have long been exploited for label-free diagnostics. While traditional approaches relied on bulk tissue or suspension measurements, advances in microfabrication have enabled single-cell electrical characterization, revealing subtle heterogeneities essential for precision medicine. Building on recent progress in single-cell impedance spectroscopy, we present a novel microfluidic impedance-based platform capable of rapid dielectric characterization of flowing cells at fourteen simultaneous, logarithmically spaced frequencies between 250 kHz and 50 MHz, achieving an unprecedented resolution of 6.1 data points per decade. The system integrates two synchronized lock-in amplifiers and a tailored signal-processing algorithm to reconstruct high-resolution impedance spectra at a throughput of up to 40 spectra per second. We demonstrate the approach on red blood cells (RBCs) and yeast cells, both under native conditions and under stress. For RBCs, chemical stress induced by antimicrobial peptides (AMPs) DNS-PMAP23 and trichogin GA IV revealed distinct dielectric alterations, while yeast cells exhibited measurable changes under thermal stress. Thousands of spectra were acquired and fitted to physically grounded models, enabling extraction of intrinsic parameters such as membrane capacitance and intracellular conductivity, as well as cell size. This work demonstrates the feasibility of high-throughput, high-resolution impedance spectroscopy of single cells, opening avenues for advanced diagnostics and biotechnological applications.

Fluorescence‐based methods are widely used to assess protein–ligand and protein–protein interactions. Thanks to their high sensitivity, these techniques allow very low dissociation constants to be determined. However, in some cases, results must be critically revised because some artifacts can occur, one of the most common being the inner filter effect (IFE). In this article, we show how multiple strategies should be used to correct data obtained from fluorescence quenching experiments to avoid overestimation of binding affinities. The extent of the proposed corrections will depend on the spectroscopic properties of the protein and the ligand, i.e., the fluorophore involved in the experiments, as well as on the specific experimental conditions in each case.

Antimicrobial peptides are promising agents for combating resistant infections. They exhibit bactericidal activity against a wide range of microbes, primarily by disrupting the permeability of the bacterial membrane and ultimately causing cell death. Effective bacterial killing requires a high number of membrane-bound peptide molecules. Therefore, it is conceivable that peptide accumulation on the membrane could also interfere with essential cellular processes by altering bilayer dynamics, a hypothesis referred to as the “sand in the gearbox” model. In this work, we systematically investigated how membrane dynamics is affected by a set of well-characterized yet highly diverse peptides: the natural AMP magainin 2, the toxin melittin, the synthetic peptides LAH4 and Killer-FLIP, and small membrane-active peptidomimetics with bactericidal activity. These effects were examined using fluorescence spectroscopy techniques, by measuring anisotropy, generalized polarization, and excimer formation of specific probes inserted at different depths within the lipid bilayer. Our results show that the activity of all compounds extends beyond membrane permeabilization, and that perturbation of membrane dynamics is a common feature among all systems analyzed. The membrane-active compounds induced a stiffening of the phospholipid bilayer by reducing lipid lateral mobility and decreasing water penetration, at least on the nanosecond timescale accessible to fluorescence measurements. Interestingly, when accounting for differences in the resulting membrane surface area coverage, the concentration range in which this behavior occurred was the same for all compounds studied. This threshold is, generally, higher than that required for membrane permeabilization and reflects near-complete coverage of the bilayer surface.

Background/Objectives: Antimicrobial peptides are promising agents for combating resistant infections. They exhibit bactericidal activity against a wide range of microbes, primarily by disrupting the permeability of the bacterial membrane and ultimately causing cell death. Effective bacterial killing requires a high number of membrane-bound peptide molecules. Therefore, it is conceivable that peptide accumulation on the membrane could also interfere with essential cellular processes by altering bilayer dynamics, a hypothesis referred to as the “sand-in-a-gearbox” model. Methods: We systematically investigated how membrane dynamics is affected by a set of well-characterized yet highly diverse peptides: the natural AMP magainin 2, the toxin melittin, the synthetic peptides LAH4 and Killer-FLIP, and small membrane-active peptidomimetics with bactericidal activity. These effects were examined using fluorescence spectroscopy techniques, by measuring anisotropy, generalized polarization, and excimer formation of specific probes inserted at different depths within the lipid bilayer. Results: The activity of all compounds extends beyond membrane permeabilization, and the perturbation of membrane dynamics, often associated with the so-called “sand-in-a-gearbox” mechanism, is a common feature among all systems analyzed. The membrane-active compounds induced a stiffening of the phospholipid bilayer by reducing lipid lateral mobility and decreasing water penetration, at least on the nanosecond timescale accessible to fluorescence measurements. Conclusions: The concentration range in which this behavior occurred was the same for all compounds studied. This threshold is, generally, higher than that required for membrane permeabilization and reflects near-complete coverage of the bilayer surface.