Storage conditions determine the characteristics of red blood cell derived extracellular vesicles.

Extracellular vesicles (EVs) are released during the storage of red blood cell (RBC) concentrates and might play adverse or beneficial roles throughout the utilization of blood products (transfusion). Knowledge of EV release associated factors and mechanism amends blood product management. In the present work the impact of storage time and medium (blood preserving additive vs isotonic phosphate buffer) on the composition, size, and concentration of EVs was studied using attenuated total reflection infrared (ATR-IR) spectroscopy, microfluidic resistive pulse sensing (MRPS) and freeze-fraction combined transmission electron micrography (FF-TEM). The spectroscopic protein-to-lipid ratio based on amide and the C-H stretching band intensity ratio indicated the formation of various vesicle subpopulations depending on storage conditions. After short storage, nanoparticles with high relative protein content were detected. Spectral analysis also suggested differences in lipid and protein composition, too. The fingerprint region (from 1300 to 1000 cm-1) of the IR spectra furnishes additional information about the biomolecular composition of RBC-derived EVs (REVs) such as adenosine triphosphate (ATP), lactose, glucose, and oxidized hemoglobin. The difference between the vesicle subpopulations reveals the complexity of the REV formation mechanism. IR spectroscopy, as a quick, cost-effective, and label-free technique provides valuable novel biochemical insight and might be used complementary to traditional omics approaches on EVs.

Quorum Sensing Pseudomonas Quinolone Signal Forms Chiral Supramolecular Assemblies With the Host Defense Peptide LL-37.

Host defense antimicrobial peptides (HDPs) constitute an integral component of the innate immune system having nonspecific activity against a broad spectrum of microorganisms. They also have diverse biological functions in wound healing, angiogenesis, and immunomodulation, where it has also been demonstrated that they have a high affinity to interact with human lipid signaling molecules. Within bacterial biofilms, quorum sensing (QS), the vital bacterial cell-to-cell communication system, is maintained by similar diffusible small molecules which control phenotypic traits, virulence factors, biofilm formation, and dispersion. Efficient eradication of bacterial biofilms is of particular importance as these colonies greatly help individual cells to tolerate antibiotics and develop antimicrobial resistance. Regarding the antibacterial function, for several HDPs, including the human cathelicidin LL-37, affinity to eradicate biofilms can exceed their activity to kill individual bacteria. However, related underlying molecular mechanisms have not been explored yet. Here, we employed circular dichroism (CD) and UV/VIS spectroscopic analysis, which revealed that LL-37 exhibits QS signal affinity. This archetypal representative of HDPs interacts with the Pseudomonas quinolone signal (PQS) molecules, producing co-assemblies with peculiar optical activity. The binding of PQS onto the asymmetric peptide chains results in chiral supramolecular architectures consisting of helically disposed, J-aggregated molecules. Besides the well-known bacterial membrane disruption activity, our data propose a novel action mechanism of LL-37. As a specific case of the so-called quorum quenching, QS signal molecules captured by the peptide are sequestered inside co-assemblies, which may interfere with the microbial QS network helping to prevent and eradicate bacterial infections.

Structural Water Stabilizes Protein Motifs in Liquid Protein Phase: The Folding Mechanism of Short ß-Sheets Coupled to Phase Transition.

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.

New short cationic antibacterial peptides. Synthesis, biological activity and mechanism of action.

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.

Role of oligo(malic acid) on the formation of unilamellar vesicles.

Stable unilamellar dipalmitoylphosphatidylcholine vesicles were produced by using oligo(malic acid) and cholesterol. Detailed physico-chemical characterization prove that by using oligo(malic acid) the substitution of PEGylated lipids for sterically stabilization comes possible. The polymer molecules cover the outer surface of spherical-shaped vesicles, and an asymmetrical composition occurs in the two leaflets of the phospholipid bilayer. The oligo(malic-acid) and cholesterol are enriched in the outer side assuring the stabilization of vesicles. Cholesterol plays an important role in the self-assembly of components as it makes the entering of oligomers possible deep into the polar head-region of lipids. The presence of oligo(malic acid) molecules does not induce degradation by hydrolysis of lipid molecules but the vesicle system turns into a sensitive form giving a possibility for pH sensitive targeting. Preliminary investigation on the investigated oligo(malic acid)-stabilized vesicles do not show any toxic effect promising their applicability in the field of liposomal drug delivery.

Characterization of extracellular vesicles by IR spectroscopy: Fast and simple classification based on amide and CH stretching vibrations.

Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investigated by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Amide and CH stretching band intensity ratios calculated from IR bands, characteristic of protein and lipid components, proved to be distinctive for the different extracellular vesicle subpopulations. This proposed 'spectroscopic protein-to-lipid ratio', combined with the outlined spectrum-analysis protocol is valid also for low sample concentrations (0.15-0.05mg/ml total protein content) and can carry information about the presence of other non-vesicular formations such as aggregated proteins, lipoproteins and immune complexes. Detailed analysis of IR data reveals compositional changes of extracellular vesicles subpopulations: second derivative spectra suggest changes in protein composition from parent cell towards exosomes favoring proteins with ß-turns and unordered motifs at the expense of intermolecular ß-sheet structures. The IR-based protein-to-lipid assessment protocol was tested also for red blood cell derived microvesicles for which similar values were obtained. The potential applicability of this technique for fast and efficient characterization of vesicular components is high as the investigated samples require no further preparations and all the different molecular species can be determined in the same sample. The results indicate that ATR-FTIR measurements provide a simple and reproducible method for the screening of extracellular vesicle preparations. It is hoped that this sophisticated technique will have further impact in extracellular vesicle research.

Preparation, purification, and characterization of aminopropyl-functionalized silica sol.

A new, simple, and "green" method was developed for the surface modification of 20 nm diameter Stöber silica particles with 3-aminopropyl(diethoxy)methylsilane in ethanol. The bulk polycondensation of the reagent was inhibited and the stability of the sol preserved by adding a small amount of glacial acetic acid after appropriate reaction time. Centrifugation, ultrafiltration, and dialysis were compared in order to choose a convenient purification technique that allows the separation of unreacted silylating agent from the nanoparticles without destabilizing the sol. The exchange of the solvent to acidic water during the purification yielded a stable colloid, as well. Structural and morphological analysis of the obtained aminopropyl silica was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements, Fourier-transform infrared (FTIR), (13)C and (29)Si MAS nuclear magnetic resonance (NMR) spectroscopies, as well as small angle X-ray scattering (SAXS). Our investigations revealed that the silica nanoparticle surfaces were partially covered with aminopropyl groups, and multilayer adsorption followed by polycondensation of the silylating reagent was successfully avoided. The resulting stable aminopropyl silica sol (ethanolic or aqueous) is suitable for biomedical uses due to its purity.