Against the increase of bacterial resistance to traditional antibiotics, antimicrobial peptides (AMP) are considered as promising alternatives. Bacterial biofilms are more resistant to antibiotics ...that their planktonic counterpart. The purpose of this study was to investigate the action of an AMP against a nascent bacterial biofilm. The activity of dermaseptin S4 derivative S4(1–16)M4Ka against 6h-old Pseudomonas fluorescens biofilms was assessed by using a combination of Attenuated Total Reflectance–Fourier Transform InfraRed (ATR–FTIR) spectroscopy in situ and in real time, fluorescence microscopy using the Baclight™ kit, and Atomic Force Microscopy (AFM, imaging and force spectroscopy). After exposure to the peptide at three concentrations, different dramatic and fast changes over time were observed in the ATR–FTIR fingerprints reflecting a concentration-dependent action of the AMP. The ATR–FTIR spectra revealed major biochemical and physiological changes, adsorption/accumulation of the AMP on the bacteria, loss of membrane lipids, bacterial detachment, bacterial regrowth, or inhibition of biofilm growth. AFM allowed estimating at the nanoscale the effect of the AMP on the nanomechanical properties of the sessile bacteria. The bacterial membrane elasticity data measured by force spectroscopy were consistent with ATR–FTIR spectra, and they allowed suggesting a mechanism of action of this AMP on sessile P. fluorescens. The combination of these three techniques is a powerful tool for in situ and in real time monitoring the activity of AMPs against bacteria in a biofilm.
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•In situ investigation of action of a dermaseptin derivative on a bacterial biofilm•Dramatic and fast changes over time were observed in the ATR–FTIR fingerprints.•AFM allowed estimating bacterial membrane elasticities without and with the peptide.•Concentration-dependent mechanisms are suggested from complementary methods.
Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in ...beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.
The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Here, we show that single-molecule force spectroscopy is a valuable tool for ...the localization and conformational analysis of individual polysaccharides on live bacteria. We focus on the clinically important probiotic bacterium Lactobacillus rhamnosus GG, demonstrating the power of AFM to reveal the coexistence of polysaccharide chains of different nature on the cell surface. Applicable to a wide variety of cells, this single molecule method offers exciting prospects for analyzing the heterogeneity and diversity of macromolecules constituting cell membranes and cell walls.
Breast cancer is the leading cause of death from cancer among women. Higher consumption of dietary marine n-3 long-chain polyunsaturated fatty acids (LC-PUFAs) is associated with a lower ...risk of breast cancer. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are two n-3 LC-PUFAs found in fish and exert anticancer effects. In this study, natural marine-derived lecithin that is rich in various polyunsaturated fatty acids (PUFAs) was extracted from salmon heads and transformed into nanoliposomes. These nanoliposomes were characterized and cultured with two breast cancer lines (MCF-7 and MDA-MB-231). The nanoliposomes decreased the proliferation and the stiffness of both cancer cell types. These results suggest that marine-derived lecithin possesses anticancer properties, which may have an impact on developing new liposomal delivery strategies for breast cancer treatment.
Despite the exceptional progress in breast cancer pathogenesis, prognosis, diagnosis, and treatment strategies, it remains a prominent cause of female mortality worldwide. Additionally, although ...chemotherapies are effective, they are associated with critical limitations, most notably their lack of specificity resulting in systemic toxicity and the eventual development of multi-drug resistance (MDR) cancer cells. Liposomes have proven to be an invaluable drug delivery system but of the multitudes of liposomal systems developed every year only a few have been approved for clinical use, none of which employ active targeting. In this review, we summarize the most recent strategies in development for actively targeted liposomal drug delivery systems for surface, transmembrane and internal cell receptors, enzymes, direct cell targeting and dual-targeting of breast cancer and breast cancer-associated cells, e.g., cancer stem cells, cells associated with the tumor microenvironment, etc.
The latest strategies for the active targeting of breast cancer and breast cancer-associated cells using surface, transmembrane and internal cell receptors, enzymes, direct cell targeting and dual-targeting liposomal drug delivery nanocarriers are systematically summarized. This paper presents the non-targeted liposomes currently in clinical use for breast cancer treatment, as well as the targeted liposomes which have progressed to clinical trials. The major challenges in the development of actively targeted drug delivery systems for cancer is also highlighted. Display omitted
•Breast cancer is the most diagnosed cancer for women worldwide.•Liposomes are immensely versatile and advantageous drug delivery tools.•Only four liposome-based treatments are in clinical use for breast cancer treatment and none make use of a targeting system.•Targeting liposome-based systems include surface, transmembrane and internal cell receptors, enzymes, and dual-targeting of breast cancer cells.
This study aims to design an optimal polyelectrolyte multilayer film of poly-l-lysine (PLL) and hyaluronic acid (HA) as an anti-inflammatory cytokine release system in order to decrease the implant ...failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1β). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties.
Deciphering the mechanism of Alzheimer’s disease is a key element for designing an efficient therapeutic strategy. Molecular dynamics (MD) calculations, atomic force microscopy, and infrared ...spectroscopy were combined to investigate β-amyloid (Aβ1–42) peptide interactions with supported lipid bilayers (SLBs). The MD simulations showed that nascent Aβ1–42 monomers remain anchored within a model phospholipid bilayer’s hydrophobic core, which suggests their stability in their native environment. We tested this prediction experimentally by studying the behavior of Aβ1–42 monomers and oligomers when interacting with SLBs. When Aβ1–42 monomers and oligomers were self-assembled with a lipid bilayer and deposited as an SLB, they remain within the bilayers. Their presence in the bilayers induces destabilization of the model membranes. No specific interactions between Aβ1–42 and the SLBs were detected when SLBs free of Aβ1–42 were exposed to Aβ1–42. This study suggests that Aβ can remain in the membrane after cleavage by γ-secretase and cause severe damage to the membrane.
Atomic force microscopy (AFM) has now become a powerful technique for investigating on a molecular level, surface forces, nanomechanical properties of deformable particles, biomolecular interactions, ...kinetics, and dynamic processes. This paper specifically focuses on the analysis of AFM force curves collected on biological systems, in particular, bacteria. The goal is to provide fully automated tools to achieve theoretical interpretation of force curves on the basis of adequate, available physical models. In this respect, we propose two algorithms, one for the processing of approach force curves and another for the quantitative analysis of retraction force curves. In the former, electrostatic interactions prior to contact between AFM probe and bacterium are accounted for and mechanical interactions operating after contact are described in terms of Hertz-Hooke formalism. Retraction force curves are analyzed on the basis of the Freely Jointed Chain model. For both algorithms, the quantitative reconstruction of force curves is based on the robust detection of critical points (jumps, changes of slope or changes of curvature) which mark the transitions between the various relevant interactions taking place between the AFM tip and the studied sample during approach and retraction. Once the key regions of separation distance and indentation are detected, the physical parameters describing the relevant interactions operating in these regions are extracted making use of regression procedure for fitting experiments to theory. The flexibility, accuracy and strength of the algorithms are illustrated with the processing of two force-volume images, which collect a large set of approach and retraction curves measured on a single biological surface. For each force-volume image, several maps are generated, representing the spatial distribution of the searched physical parameters as estimated for each pixel of the force-volume image.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Metal-based complexes are well-established cancer chemotherapeutic drug candidates. Although our knowledge regarding their exact activity
vs.
toxicity profile is incomplete, changes in cell membrane ...biophysical properties and cytoskeletal structures have been implicated as part of the mechanism of action. Thus, in this work, we characterised the effects of iron(
ii
)-based complexes on the structural and morphological properties of epithelial non-tumorigenic (MCF 10A) and tumorigenic (MDA-MB-231) breast cell lines using atomic force microscopy (AFM), flow cytometry and immunofluorescence microscopy. At 24 h of exposure, both the MCF 10A and MDA-MB-231 cells experienced a cell softening, and an increase in size followed by a re-stiffening at 96 h. In addition, the triple negative breast cancer cell line, MDA-MB-231, sustained a notable cytoskeletal and mitochondrial reorganization with increased actin stress fibers and cell-to-cell communication structures. An extensive all-atom molecular dynamic simulation suggests a possible direct and unassisted internalization of the metallodrug candidate, and confirmed that the cellular effects could not be ascribed to the simple physical interaction of the iron-based complexes with the biological membrane. These observations provide an insight into a link between the mechanisms of action of such iron-based complexes as anti-cancer treatment and cytoskeletal architecture.
Iron(
ii
)-based complexes were able to penetrate into the cytoplasm without disrupting the cell membrane. The complexes induced size, morphological, stiffness and cytoarchitectural changes in both triple negative breast cancer and normal cells.
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