Development of a New Binary Matrix for the Comprehensive Analysis of Lipids and Pigments in Micro- and Macroalgae Using MALDI-ToF/ToF Mass Spectrometry.

While edible algae might seem low in fat, the lipids they contain are crucial for good health and preventing chronic diseases. This study introduces a binary matrix to analyze all the polar lipids in both macroalgae (Wakame-Undaria pinnatifida, Dulse-Palmaria palmata, and Nori-Porphyra spp.) and microalgae (Spirulina-Arthrospira platensis, and Chlorella-Chlorella vulgaris) using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The key lies in a new dual matrix made by combining equimolar amounts of 1,5-diaminonaphthalene (DAN) and 9-aminoacridine (9AA). This combination solves the limitations of single matrices: 9AA is suitable for sulfur-containing lipids and acidic phospholipids, while DAN excels as an electron-transfer secondary reaction matrix for intact chlorophylls and their derivatives. By employing the equimolar binary matrix, a wider range of algal lipids, including free fatty acids, phospholipids, glycolipids, pigments, and even rare arsenosugarphospholipids were successfully detected, overcoming drawbacks related to ion suppression from readily ionizable lipids. The resulting mass spectra exhibited a good signal-to-noise ratio at a lower laser fluence and minimized background noise. This improvement stems from the binary matrix's ability to mitigate in-source decay effects, a phenomenon often encountered for certain matrices. Consequently, the data obtained are more reliable, facilitating a faster and more comprehensive exploration of algal lipidomes using high-throughput MALDI-MS/MS analysis.

Effect of osmotic stress on live cell plasma membranes, probed via Laurdan general polarization measurements.

Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdan's generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress.

The highly packed and dehydrated structure of preformed unexposed human pulmonary surfactant isolated from amniotic fluid.

By coating the alveolar air-liquid interface, lung surfactant overwhelms surface tension forces that, otherwise, would hinder the lifetime effort of breathing. Years of research have provided a picture of how highly hydrophobic and specialized proteins in surfactant promote rapid and efficient formation of phospholipid-based complex three-dimensional films at the respiratory surface, highly stable under the demanding breathing mechanics. However, recent evidence suggests that the structure and performance of surfactant typically isolated from bronchoalveolar lung lavages may be far from that of nascent, still unused, surfactant as freshly secreted by type II pneumocytes into the alveolar airspaces. In the present work, we report the isolation of lung surfactant from human amniotic fluid (amniotic fluid surfactant, AFS) and a detailed description of its composition, structure, and surface activity in comparison to a natural surfactant (NS) purified from porcine bronchoalveolar lavages. We observe that the lipid/protein complexes in AFS exhibit a substantially higher lipid packing and dehydration than in NS. AFS shows melting transitions at higher temperatures than NS and a conspicuous presence of nonlamellar phases. The surface activity of AFS is not only comparable with that of NS under physiologically meaningful conditions but displays significantly higher resistance to inhibition by serum or meconium, agents that inactivate surfactant in the context of severe respiratory pathologies. We propose that AFS may be the optimal model to study the molecular mechanisms sustaining pulmonary surfactant performance in health and disease, and the reference material to develop improved therapeutic surfactant preparations to treat yet unresolved respiratory pathologies.

LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity.

Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.

Signal enhancement of hyperpolarized 15 N sites in solution-increase in solid-state polarization at 3.35 T and prolongation of relaxation in deuterated water mixtures.

Hyperpolarized 15 N sites have been found to be promising for generating long-lived hyperpolarized states in solution, and present a promising approach for utilizing dissolution-dynamic nuclear polarization (dDNP)-driven hyperpolarized MRI for imaging in biology and medicine. Specifically, 15 N sites with directly bound protons were shown to be useful when dissolved in D2 O. The purpose of the current study was to further characterize and increase the visibility of such 15 N sites in solutions that mimic an intravenous injection during the first cardiac pass in terms of their H2 O:D2 O composition. The T1 values of hyperpolarized 15 N in [15 N2 ]urea and [15 N]NH4 Cl demonstrated similar dependences on the H2 O:D2 O composition of the solution, with a T1 of about 140 s in 100% D2 O, about twofold shortening in 90% and 80% D2 O, and about threefold shortening in 50% D2 O. [13 C]urea was found to be a useful solid-state 13 C marker for qualitative monitoring of the 15 N polarization process in a commercial pre-clinical dDNP device. Adding trace amounts of Gd3+ to the polarization formulation led to higher solid-state polarization of [13 C]urea and to higher polarization levels of [15 N2 ]urea in solution.

Evaluating membrane structure by Laurdan imaging: Disruption of lipid packing by oxidized lipids.

Impact of different lipids on membrane structure/lipid order is critical for multiple biological processes. Laurdan microscopy provides a unique tool to assess this property in heterogeneous biological membranes. This review describes the general principles of the approach and its application in model membranes and cells. It also provides an in-depth discussion of the insights obtained using Laurdan microscopy to evaluate the differential effects of cholesterol, oxysterols and oxidized phospholipids on lipid packing of ordered and disordered domains in vascular endothelial cells.

PAD4 inhibitor promotes DNA damage and radiosensitivity of nasopharyngeal carcinoma cells.

Peptidylarginine deiminases 4 (PAD4), a kind of enzyme capable of converting protein arginine or mono-methylarginine into citrulline, has been identified to display a key role in diverse diseases. Radiotherapy is frequently used in nasopharyngeal carcinoma (NPC) treatment and induces DNA double strand breaks. In this study, whether PAD4 inhibitor YW3-56 affects the radiosensitivity of NPC cells was explored. RT-qPCR, immunofluorescence, western blot, clonogenic survival, and flow cytometry assays were used to assess the function of PAD4 and YW3-56 in NPC. We found the upregulation of PAD4 expression in NPC cells. PAD4 overexpression suppressed NPC cell apoptosis and promoted cell cycle, while PAD4 depletion had an opposite result. Moreover, the survival of NPC cells after irradiation was increased by overexpression of PAD4. PAD4 overexpression inhibited DNA damage and sensitivity of NPC cells to irradiation. Functional assays showed that YW3-56 treatment promoted DNA damage, apoptosis, and radiosensitivity of NPC cells. Importantly, YW3-56 treatment inhibited tumor growth in vivo. Overall, this study revealed the efficacy of PAD4 inhibitor YW3-56 in promoting sensitivity of NPC cells to irradiation.

Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

Photophysical Properties of BADAN Revealed in the Study of GGBP Structural Transitions.

The fluorescent dye BADAN (6-bromoacetyl-2-dimetylaminonaphtalene) is widely used in various fields of life sciences, however, the photophysical properties of BADAN are not fully understood. The study of the spectral properties of BADAN attached to a number of mutant forms of GGBP, as well as changes in its spectral characteristics during structural changes in proteins, allowed to shed light on the photophysical properties of BADAN. It was shown that spectral properties of BADAN are determined by at least one non-fluorescent and two fluorescent isomers with overlapping absorbing bands. It was found that BADAN fluorescence is determined by the unsolvated "PICT" (planar intramolecular charge transfer state) and solvated "TICT" (twisted intramolecular charge transfer state) excited states. While "TICT" state can be formed both as a result of the "PICT" state solvation and as a result of light absorption by the solvated ground state of the dye. BADAN fluorescence linked to GGBP/H152C apoform is quenched by Trp 183, but this effect is inhibited by glucose intercalation. New details of the changes in the spectral characteristics of BADAN during the unfolding of the protein apo and holoforms have been obtained.

Redesigning Solvatochromic Probe Laurdan for Imaging Lipid Order Selectively in Cell Plasma Membranes.

Imaging of biological membranes by environmentally sensitive solvatochromic probes, such as Laurdan, provides information about the organization of lipids, their ordering, and their uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labeling of internal membranes, we redesigned it by introducing a membrane anchor group based on negatively charged sulfonate and dodecyl chain. The obtained probe, Pro12A, stains exclusively the outer leaflet of lipid bilayers of liposomes, as evidenced by leaflet-specific fluorescence quenching with a viologen derivative, and shows higher fluorescence brightness than Laurdan. Pro12A also exhibits stronger spectral change between liquid-ordered and liquid-disordered phases in model membranes and distinguishes better lipid domains in giant plasma membrane vesicles (GPMVs) than Laurdan. In live cells, it stains exclusively the cell plasma membranes, in contrast to Laurdan and its carboxylate analogue C-Laurdan. Owing to its outer leaflet binding, Pro12A is much more sensitive to cholesterol extraction than Laurdan, which is redistributed within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures the absence of crosstalk with a number of orange/red fluorescent proteins and dyes. Thus, Pro12A will enable accurate multicolor imaging of lipid organization of cell plasma membranes in the presence of fluorescently tagged proteins of interest, which will open new opportunities in biomembrane research.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z' ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH3 fragment ions ("twins"). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH3 ions requires consideration for accurate and confident assignments of z' ions in MALDI-ISD FT-ICR mass spectra.

An experimental system for real-time fluorescence recordings of cell membrane changes induced by electroporation.

The electroporation of cells is nowadays used for a large variety of purposes, from basic research to cancer therapy and food processing. Understanding molecular mechanisms of the main processes involved in electroporation is thus of significant interest. In the present work, we propose an experimental system to record in real time the evolution of any cell parameter which can be evaluated by fluorescence (before, during and after application of the electroporation pulses to cells in suspension). The system is based on the design of adequate electroporation electrodes, compatible with a standard spectrofluorometer cuvette housing. The electric field intensity generated when pulses are applied was carefully characterized for different geometries of the electrodes, to choose a construction ensuring the greatest homogeneity of the field in combination with the best possible illumination of the sample. As an example of the method's application, we present here generalized polarization kinetics for a varying number of electroporation pulses applied to a cell suspension; the general polarization parameter is strongly correlated to water presence in the hydrophobic membrane core. The system may be used for many other fluorescence measurements useful for the characterization of the electroporation process.

Nanodisc self-assembly is thermodynamically reversible and controllable.

Many highly ordered complex systems form by the spontaneous self-assembly of simpler subunits. An important biophysical tool that relies on self-assembly is the Nanodisc system, which finds extensive use as native-like environments for studying membrane proteins. Nanodiscs are self-assembled from detergent-solubilized mixtures of phospholipids and engineered helical proteins called membrane scaffold proteins (MSPs). Detergent removal results in the formation of nanoscale bilayers stabilized by two MSP "belts." Despite their numerous applications in biology, and contributions from many laboratories world-wide, little is known about the self-assembly process such as when the bilayer forms or when the MSP associates with lipids. We use fluorescence and optical spectroscopy to probe self-assembly at various equilibria defined by the detergent concentration. We show that the bilayer begins forming below the critical micellar concentration of the detergent (10 mM), and the association of MSP and lipids begins at lower detergent levels, showing a dependence on the concentrations of MSP and lipids. Following the dissolution process by adding detergent to purified Nanodiscs demonstrates that the self-assembly is reversible. Our data demonstrate that Nanodisc self-assembly is experimentally accessible, and that controlling the detergent concentration allows exquisite control over the self-assembly reaction. This improved understanding of self-assembly could lead to better functional incorporation of hitherto intractable membrane target proteins.

Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors.

We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.

Solvatochromic Modeling of Laurdan for Multiple Polarity Analysis of Dihydrosphingomyelin Bilayer.

The hydration properties of the interface between lipid bilayers and bulk water are important for determining membrane characteristics. Here, the emission properties of a solvent-sensitive fluorescence probe, 6-lauroyl-2-dimethylamino naphthalene (Laurdan), were evaluated in lipid bilayer systems composed of the sphingolipids D-erythro-N-palmitoyl-sphingosylphosphorylcholine (PSM) and D-erythro-N-palmitoyl-dihydrosphingomyelin (DHPSM). The glycerophospholipids 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine and 1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine were used as controls. The fluorescence properties of Laurdan in sphingolipid bilayers indicated multiple excited states according to the results obtained from the emission spectra, fluorescence anisotropy, and the center-of-mass spectra during the decay time. Deconvolution of the Laurdan emission spectra into four components based on the solvent model enabled us to identify the varieties of hydration and the configurational states derived from intermolecular hydrogen bonding in sphingolipids. Sphingolipids showed specific, interfacial hydration properties stemming from their intra- and intermolecular hydrogen bonds. Particularly, the Laurdan in DHPSM revealed more hydrated properties compared to PSM, even though DHPSM has a higher Tm than PSM. Because DHPSM forms hydrogen bonds with water molecules (in 2NH configurational functional groups), the interfacial region of the DHPSM bilayer was expected to be in a highly polar environment. The careful analysis of Laurdan emission spectra through the four-component deconvolution in this study provides important insights for understanding the multiple polarity in the lipid membrane.

Higher-energy collision-induced dissociation for the quantification by liquid chromatography/tandem ion trap mass spectrometry of nitric oxide metabolites coming from S-nitroso-glutathione in an in vitro model of the intestinal barrier.

RATIONALE: The potency of S-nitrosoglutathione (GSNO) as a nitric oxide (NO) donor to treat cardiovascular diseases (CVDs) has been highlighted in numerous studies. In order to study its bioavailability after oral administration, which represents the most convenient route for the chronic treatment of CVDs, it is essential to develop an analytical method permitting (i) the simultaneous measurement of GSNO metabolites, i.e. nitrite, S-nitrosothiols (RSNOs) and nitrate and (ii) to distinguish them from other sources (endogenous synthesis and diet). METHODS: Exogenous GSNO was labeled with 15 N, and the GS15 NO metabolites after conversion into the nitrite ion were derivatized with 2,3-diaminonaphthalene. The resulting 2,3-naphthotriazole was quantified by liquid chromatography/tandem ion trap mass spectrometry (LC/ITMS/MS) in multiple reaction monitoring mode after Higher-energy Collision-induced Dissociation (HCD). Finally, the validated method was applied to an in vitro model of the intestinal barrier (monolayer of Caco-2 cells) to study GS15 NO intestinal permeability. RESULTS: A LC/ITMS/MS method based on an original transition (m/z 171 to 156) for sodium 15 N-nitrite, GS15 NO and sodium 15 N-nitrate measurements was validated, with recoveries of 100.8 ± 3.8, 98.0 ± 2.7 and 104.1 ± 3.3%, respectively. Intra- and inter-day variabilities were below 13.4 and 12.6%, and the limit of quantification reached 5 nM (signal over blank = 4). The permeability of labeled GS15 NO (10-100 µM) was evaluated by calculating its apparent permeability coefficient (Papp ). CONCLUSIONS: A quantitative LC/ITMS/MS method using HCD was developed for the first time to selectively monitor GS15 NO metabolites. The assay allowed evaluation of GS15 NO intestinal permeability and situated this drug candidate within the middle permeability class according to FDA guidelines. In addition, the present method has opened the perspective of a more fundamental work aiming at studying the fragmentation mechanism leading to the ion at m/z 156 in HCD tandem mass spectrometry in the presence of acetonitrile.

Improved matrix coating for positive- and negative-ion-mode MALDI-TOF imaging of lipids in blood vessel tissues.

High-quality matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) of lipids in biological tissue relies on the fabrication of a homogeneous matrix coating featuring best possible analyte integration. This communication addresses a matrix vapor deposition/recrystallization process for the application of 1,5-diaminonaphthalene (1,5-DAN) onto slices of human aortic tissue. The matrix coating is compatible with both positive- as well as negative-ion-mode MALDI MSI facilitating a significantly enhanced detection of lipid-related signals in different cell layers of blood vessel walls. Graphical abstract.

Scanning fluorescence correlation spectroscopy comes full circle.

In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated.

Time-resolved Fluorescence and Generalized Polarization: Innovative tools to assess bull sperm membrane dynamics during slow freezing.

During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing).

Intragenic Antimicrobial Peptide Hs02 Hampers the Proliferation of Single- and Dual-Species Biofilms of P. aeruginosa and S. aureus: A Promising Agent for Mitigation of Biofilm-Associated Infections.

Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in a large variety of infections. Their co-occurrence in the same site of infection has been frequently reported and is linked to enhanced virulence and difficulty of treatment. Herein, the antimicrobial and antibiofilm activities of an intragenic antimicrobial peptide (IAP), named Hs02, which was uncovered from the human unconventional myosin 1H protein, were investigated against several P. aeruginosa and S. aureus strains, including multidrug-resistant (MDR) isolates. The antibiofilm activity was evaluated on single- and dual-species biofilms of P. aeruginosa and S. aureus. Moreover, the effect of peptide Hs02 on the membrane fluidity of the strains was assessed through Laurdan generalized polarization (GP). Minimum inhibitory concentration (MIC) values of peptide Hs02 ranged from 2 to 16 µg/mL against all strains and MDR isolates. Though Hs02 was not able to hamper biofilm formation by some strains at sub-MIC values, it clearly affected 24 h preformed biofilms, especially by reducing the viability of the bacterial cells within the single- and dual-species biofilms, as shown by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) images. Laurdan GP values showed that Hs02 induces membrane rigidification in both P. aeruginosa and S. aureus. Peptide Hs02 can potentially be a lead for further improvement as an antibiofilm agent.