In Vitro Effects of Postmetabolites from Limosilactobacillus fermentum 53 on the Survival and Proliferation of HT-29 Cells.

Naturally fermented dairy products are an important component of the human diet. They are a valuable source of nutrients as well as vitamins and minerals. Their importance as a source of probiotic bacterial strains should not be overlooked. A number of studies highlight the positive effects of species of the probiotic lactic acid bacteria on the intestinal microbiome and the overall homeostasis of the body, as well as a complementary treatment for some diseases. However, data on the effects on the intestinal epithelial cells of postmetabolites released by probiotic bacteria are incomplete. This is likely due to the fact that these effects are species- and strain-specific. In the present study, we investigated the effects of postmetabolites produced by a pre-selected candidate probiotic strain Limosilactobacillus fermentum on HT-29 intestinal epithelial cells. Our data showed a pronounced proliferative effect, evaluated by flow cytometry, quantification of the cell population and determination of the mitotic index. This was accompanied by the stabilization of the cell monolayer, measured by an increase in TEER (transepithelial electric resistance) and the reorganization of actin filaments. The data obtained are a clear indication of the positive effects that the products secreted by L. fermentum strain 53 have on intestinal epithelial cells.

Poly(2-(dimethylamino)ethyl methacrylate)-Grafted Amphiphilic Block Copolymer Micelles Co-Loaded with Quercetin and DNA.

The synergistic effect of drug and gene delivery is expected to significantly improve cancer therapy. However, it is still challenging to design suitable nanocarriers that are able to load simultaneously anticancer drugs and nucleic acids due to their different physico-chemical properties. In the present work, an amphiphilic block copolymer comprising a biocompatible poly(ethylene glycol) (PEG) block and a multi-alkyne-functional biodegradable polycarbonate (PC) block was modified with a number of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) side chains applying the highly efficient azide-alkyne "click" chemistry reaction. The resulting cationic amphiphilic copolymer with block and graft architecture (MPEG-b-(PC-g-PDMAEMA)) self-associated in aqueous media into nanosized micelles which were loaded with the antioxidant, anti-inflammatory, and anticancer drug quercetin. The drug-loaded nanoparticles were further used to form micelleplexes in aqueous media through electrostatic interactions with DNA. The obtained nanoaggregates-empty and drug-loaded micelles as well as the micelleplexes intended for simultaneous DNA and drug codelivery-were physico-chemically characterized. Additionally, initial in vitro evaluations were performed, indicating the potential application of the novel polymer nanocarriers as drug delivery systems.

Cholesterol Alters the Phase Separation in Model Membranes Containing hBest1.

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca2+-dependent Cl- channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (Cs-1) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca2+ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔGmixπ values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔGmixπ are retained and equalized with the values of ΔGmixπ in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.

Condensing Effect of Cholesterol on hBest1/POPC and hBest1/SM Langmuir Monolayers.

Human bestrophin-1 protein (hBest1) is a transmembrane channel associated with the calcium-dependent transport of chloride ions in the retinal pigment epithelium as well as with the transport of glutamate and GABA in nerve cells. Interactions between hBest1, sphingomyelins, phosphatidylcholines and cholesterol are crucial for hBest1 association with cell membrane domains and its biological functions. As cholesterol plays a key role in the formation of lipid rafts, motional ordering of lipids and modeling/remodeling of the lateral membrane structure, we examined the effect of different cholesterol concentrations on the surface tension of hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and hBest1/SM Langmuir monolayers in the presence/absence of Ca2+ ions using surface pressure measurements and Brewster angle microscopy studies. Here, we report that cholesterol: (1) has negligible condensing effect on pure hBest1 monolayers detected mainly in the presence of Ca2+ ions, and; (2) induces a condensing effect on composite hBest1/POPC and hBest1/SM monolayers. These results offer evidence for the significance of intermolecular protein-lipid interactions for the conformational dynamics of hBest1 and its biological functions as multimeric ion channel.

Miscibility of hBest1 and sphingomyelin in surface films - A prerequisite for interaction with membrane domains.

Human bestrophin-1 (hBest1) is a transmembrane Ca2+- dependent anion channel, associated with the transport of Cl-, HCO3- ions, γ-aminobutiric acid (GABA), glutamate (Glu), and regulation of retinal homeostasis. Its mutant forms cause retinal degenerative diseases, defined as Bestrophinopathies. Using both physicochemical - surface pressure/mean molecular area (π/A) isotherms, hysteresis, compressibility moduli of hBest1/sphingomyelin (SM) monolayers, Brewster angle microscopy (BAM) studies, and biological approaches - detergent membrane fractionation, Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) and immunofluorescence staining of stably transfected MDCK-hBest1 and MDCK II cells, we report: 1) Ca2+, Glu and GABA interact with binary hBest1/SM monolayers at 35 °C, resulting in changes in hBest1 surface conformation, structure, self-organization and surface dynamics. The process of mixing in hBest1/SM monolayers is spontaneous and the effect of protein on binary films was defined as "fluidizing", hindering the phase-transition of monolayer from liquid-expanded to intermediate (LE-M) state; 2) in stably transfected MDCK-hBest1 cells, bestrophin-1 was distributed between detergent resistant (DRM) and detergent-soluble membranes (DSM) - up to 30 % and 70 %, respectively; in alive cells, hBest1 was visualized in both liquid-ordered (Lo) and liquid-disordered (Ld) fractions, quantifying protein association up to 35 % and 65 % with Lo and Ld. Our results indicate that the spontaneous miscibility of hBest1 and SM is a prerequisite to diverse protein interactions with membrane domains, different structural conformations and biological functions.