The potential of AFM in studying the role of the nanoscale amphipathic nature of (lipo)-peptides interacting with lipid bilayers.

Antimicrobial peptides (AMPs) and lipopeptides (LPs) represent very promising molecules to fight resistant bacterial infections due to their broad-spectrum of activity, their first target, i.e. the bacterial membrane, and the rapid bactericidal action. For both types of molecules, the action mechanism starts from the membrane of the pathogen agents, producing a disorganization of their phase structure or the formation of pores of different size altering their permeability. This mechanism of action is based on physical interactions more than on a lock-and-key recognition event and it is difficult for the pathogens to rapidly develop an effective resistance. Very small differences in the sequence of both AMPs and LPs might lead to very different effects on the target membrane. Therefore, a correct understanding of their mechanism of action is required with the aim of developing new synthetic peptides, analogues of the natural ones, with specific and more powerful bactericidal activity. Atomic force microscopy (AFM), with its high resolution and the associated force spectroscopy resource, provides a valuable technique to investigate the reorganization of lipid bilayers exposed to antimicrobial or lipopeptides. Here, we present AFM results obtained by ours and other groups on the action of AMPs and LPs on supported lipid bilayers (SLBs) of different composition. We also consider data obtained by fluorescence microscopy to compare the AFM data with another technique which can be used on different lipid bilayer model systems such as SLBs and giant unilamellar vesicles. The outcomes here presented highlight the powerful of AFM-based techniques in detecting nanoscale peptide-membrane interactions and strengthen their use as an exceptional complementary tool toin vivoinvestigations. Indeed, the combination of these approaches can help decipher the mechanisms of action of different antimicrobials and lipopeptides at both the micro and nanoscale levels, and to design new and more efficient antimicrobial compounds.

Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells.

In this study, extracts from non-psychoactive Cannabis sativa L. varieties were characterized by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)-rich extract inhibited cell proliferation of K562 cell line in a dose-dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

An Overview on Microfluidic Systems for Nucleic Acids Extraction from Human Raw Samples.

Nucleic acid (NA) extraction is a basic step for genetic analysis, from scientific research to diagnostic and forensic applications. It aims at preparing samples for its application with biomolecular technologies such as isothermal and non-isothermal amplification, hybridization, electrophoresis, Sanger sequencing and next-generation sequencing. Multiple steps are involved in NA collection from raw samples, including cell separation from the rest of the specimen, cell lysis, NA isolation and release. Typically, this process needs molecular biology facilities, specialized instrumentation and labor-intensive operations. Microfluidic devices have been developed to analyze NA samples with high efficacy and sensitivity. In this context, the integration within the chip of the sample preparation phase is crucial to leverage the promise of portable, fast, user-friendly and economic point-of-care solutions. This review presents an overview of existing lab-on-a-chip (LOC) solutions designed to provide automated NA extraction from human raw biological fluids, such as whole blood, excreta (urine and feces), saliva. It mainly focuses on LOC implementation aspects, aiming to describe a detailed panorama of strategies implemented for different human raw sample preparations.

Model lipid systems and their use to evaluate the phase state of biomembranes, their mechanical properties and the effect of non-conventional antibiotics: the case of daptomycin.

The lipid bilayer is the basis of the structure and function of the cell membrane. The study of the molecular phenomena that affect biological membranes has a great impact on the understanding of cellular physiology. To understand these phenomena, it has become increasingly necessary to develop simple synthetic models that allow the most basic details of such processes to be reproduced. In this short communication, we took advantage of the properties of two well-established lipid model systems, GUVs and SLBs, with compositions mimicking the cell membrane present in mammals and bacteria, to study the thermotropic phase behavior of lipids as well as the effect of daptomycin, a cyclic lipopeptide used as an antibiotic. The study of mechanical and thermodynamical properties of these model systems could contribute to establish a theoretical framework to develop more efficient strategies for biological control.

Complex Phase Behavior of GUVs Containing Different Sphingomyelins.

Understanding the lateral organization of biological membranes plays a key role on the road to fully appreciate the physiological functions of this fundamental barrier between the inside and outside regions of a cell. Ternary lipid bilayers composed of a high and a low melting temperature lipid and cholesterol represent a model system that mimics some of the important thermodynamical features of much more complex lipid mixtures such as those found in mammal membranes. The phase diagram of these ternary mixtures can be studied exploiting fluorescence microscopy in giant unilamellar vesicles, and it is typically expected to give rise, for specific combinations of composition and temperature, to regions of two-phase coexistence and a region with three-phase coexistence, namely, the liquid-ordered, liquid-disordered, and solid phases. Whereas the observation of two-phase coexistence is routinely possible using fluorescence microscopy, the three-phase region is more elusive to study. In this article, we show that particular lipid mixtures containing diphytanoyl-phosphatidylcholine and cholesterol plus different types of sphingomyelin (SM) are prone to produce bilayer regions with more than two levels of fluorescence intensity. We found that these intensity levels occur at low temperature and are linked to the copresence of long and asymmetric chains in SMs and diphytanoyl-phosphatidylcholine in the lipid mixtures. We discuss the possible interpretations for this observation in terms of bilayer phase organization in the presence of sphingolipids. Additionally, we also show that in some cases, liposomes in the three-phase coexistence state exhibit extreme sensitivity to lateral tension. We hypothesize that the appearance of the different phases is related to the asymmetric structure of SMs and to interdigitation effects.

Role of Lipid Composition, Physicochemical Interactions, and Membrane Mechanics in the Molecular Actions of Microbial Cyclic Lipopeptides.

Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide-lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling-at the biophysical level-the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.

Glutamate Receptors and Glioblastoma Multiforme: An Old "Route" for New Perspectives.

Glioblastoma multiforme (GBM) is the most aggressive malignant tumor of the central nervous system, with poor survival in both treated and untreated patients. Recent studies began to explain the molecular pathway, comprising the dynamic structural and mechanical changes involved in GBM. In this context, some studies showed that the human glioblastoma cells release high levels of glutamate, which regulates the proliferation and survival of neuronal progenitor cells. Considering that cancer cells possess properties in common with neural progenitor cells, it is likely that the functions of glutamate receptors may affect the growth of cancer cells and, therefore, open the road to new and more targeted therapies.

The Rejuvenating Effect and Tolerability of an Auto-Cross-Linked Hyaluronic Acid on Décolletage: A Pilot Prospective Study.

BACKGROUND: A novel auto-cross-linked hyaluronic acid formulation (HA-ACP) has been proposed as a treatment for skin rejuvenation. This prospective pilot study aims to assess its tolerability and performance in the treatment of skin aging of the décolletage. METHODS: Fifteen women (mean age 49.0 ± 8.0  (range 30-65)) who complained about the appearance of their décolletage were prospectively enrolled and treated with three sets of HA-ACP (IAL-SYSTEM ACP®) intradermal injections, administered three weeks apart. Control visits followed at 80 and 120 days from enrollment. The rejuvenating effect was assessed using the Beagley-Gibson and Fabi-Bolton scores. The investigators and the subjects also subjectively assessed skin appearance using a 1-10 scale. The rejuvenating effect and its persistence were assessed comparing the scores collected at the different experimental times by means of Wilcoxon rank tests. Adverse events and their duration were recorded and compared to those reported in the literature. RESULTS: The HA-ACP formulation provided a significant improvement according to the Beagley-Gibson score (p < 0.005) at all time points. At the 120-day follow-up, the Fabi-Bolton score was significantly improved with respect to baseline (p < 0.05). The rejuvenating effect persisted unaltered between the 80-day and the 120-day visits. At the 120-day visit, the subjects' and investigators' subjective scores were between 8 and 10 in most cases. No unexpected adverse effects were observed. All adverse effects were well tolerated and disappeared over a short period. CONCLUSIONS: The HA-ACP treatment under investigation is safe, well tolerated, and provides effective and long-lasting décolletage rejuvenation. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study.

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study.

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABAA receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50-200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABAA receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABAA receptor activity and their effects on a lipid bilayer model system containing cholesterol.

Evaluation of a new hyaluronic acid dermal filler for volume restoration.

OBJECTIVES: The present study aimed to evaluate the effectiveness and safety of hyaluronic acid gel for the treatment of volume loss or contour deficiency. METHODS: This 6-month, open-label study recruited sixty adults aged 40-65 years with bilateral moderate to severe volume loss or contour deficiency (Facial Volume Loss Scale [FVLS] score: 2-5). Eligible subjects were treated at the baseline visit with an injection of hyaluronic acid gel in one or more of three facial subregions: the zygomatic malar region, the anteromedial cheek and nasolabial folds. RESULTS: A significant improvement from baseline was observed in the FVLS score at each study visit up to 6 months post treatment (p < 0.0125 by Wilcoxon test). A significant improvement was also observed in the Global Aesthetic Improvement Scale (GAIS) at each visit up to 6 months. The study showed that the GAIS score improved in 100% of subjects at month 3, while at the end of the study (month 6) the percentage of improved subjects decreased to 91. Treatment effect was maintained by 100% of subjects up to month 3, while 57% of subjects showed a persistence of effect up to month 6. CONCLUSIONS: Volume loss or contour deficiency may be safely and effectively corrected using the new dermal filler.

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: an AFM investigation.

γ-Hemolysins are bicomponent ß-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.

Phase transitions in supported lipid bilayers studied by AFM.

We review the capabilities of Atomic Force Microscopy (AFM) in the study of phase transitions in Supported Lipid Bilayers (SLBs). AFM represents a powerful technique to cover the resolution range not available to fluorescence imaging techniques and where spectroscopic data suggest what the relevant lateral scale for domain formation might be. Phase transitions of lipid bilayers involve the formation of domains characterized by different heights with respect to the surrounding phase and are therefore easily identified by AFM in liquid solution once the bilayer is confined to a flat surface. Even if not endowed with high time resolution, AFM allows light to be shed on some aspects related to lipid phase transitions in the case of both a single lipid component and lipid mixtures containing sterols also. We discuss here the obtained results in light of the peculiarities of supported lipid bilayer model systems.

Extraction, purification and in vitro assessment of the antioxidant and anti-inflammatory activity of policosanols from non-psychoactive Cannabis sativa L.

Policosanols (PCs) are bioactive compounds extracted from different natural waxes. In this work, the purification, characterization and assessment of the antioxidant and anti-inflammatory activity was carried out on PCs from an innovative source, i.e. a waxy material from supercritical-fluid extraction (SFE) of non-psychoactive Cannabis sativa L. (hemp) inflorescences. Starting from this material, PCs were obtained by microwave-assisted trans-esterification and hydrolysis, followed by preparative liquid chromatography under normal phase conditions. The purified product was characterized using high-performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). In vitro cell-free and cell-based antioxidant and anti-inflammatory assays were then performed to assess their bioactivity. HPLC-ELSED analysis of the purified mixture from hemp wax revealed C26OH and C28OH as the main compounds. In vitro assays indicated an inhibition of intracellular reactive oxygen species (ROS) production, a reduction of nuclear factor kappa B (NF-κB) activation and of the activity of the neutrophil elastase. Immunoblotting assays allowed us to hypothesize the mechanism of action of the compounds of interest, given the higher levels of MAPK-activated protein kinase 2 (MK2) and heme oxygenase-1 (HO-1) protein expression in the PC pretreated HaCaT cells. In conclusion, even if more research is needed to unveil other molecular mechanisms involved in hemp PC activity, the results of this work suggest that these compounds may have potential for use in oxinflammation processes.

Cytochalasin B Influences Cytoskeletal Organization and Osteogenic Potential of Human Wharton's Jelly Mesenchymal Stem Cells.

Among perinatal stem cells of the umbilical cord, human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1-3 µM) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers.

The NF-Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer.

Aberrant splicing events are associated with colorectal cancer (CRC) and provide new opportunities for tumor diagnosis and treatment. The expression of the splice variants of NF-YA, the DNA binding subunit of the transcription factor NF-Y, is deregulated in multiple cancer types compared to healthy tissues. NF-YAs and NF-YAl isoforms differ in the transactivation domain, which may result in distinct transcriptional programs. In this study, we demonstrated that the NF-YAl transcript is higher in aggressive mesenchymal CRCs and predicts shorter patients' survival. In 2D and 3D conditions, CRC cells overexpressing NF-YAl (NF-YAlhigh) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAshigh, NF-YAlhigh cells show changes in the transcription of genes involved in epithelial-mesenchymal transition, extracellular matrix and cell adhesion. NF-YAl and NF-YAs bind similarly to the promoter of the E-cadherin gene, but oppositely regulate its transcription. The increased metastatic potential of NF-YAlhigh cells in vivo was confirmed in zebrafish xenografts. These results suggest that the NF-YAl splice variant could be a new CRC prognostic factor and that splice-switching strategies may reduce metastatic CRC progression.

Dynamic force spectroscopy on supported lipid bilayers: effect of temperature and sample preparation.

Biological membranes are constantly exposed to forces. The stress-strain relation in membranes determines the behavior of many integral membrane proteins or other membrane related-proteins that show a mechanosensitive behavior. Here, we studied by force spectroscopy the behavior of supported lipid bilayers (SLBs) subjected to forces perpendicular to their plane. We measured the lipid bilayer mechanical properties and the force required for the punch-through event characteristic of atomic force spectroscopy on SLBs as a function of the interleaflet coupling. We found that for an uncoupled bilayer, the overall tip penetration occurs sequentially through the two leaflets, giving rise to two penetration events. In the case of a bilayer with coupled leaflets, penetration of the atomic force microscope tip always occurred in a single step. Considering the dependence of the jump-through force value on the tip speed, we also studied the process in the context of dynamic force spectroscopy (DFS). We performed DFS experiments by changing the temperature and cantilever spring constant, and analyzed the results in the context of the developed theories for DFS. We found that experiments performed at different temperatures and with different cantilever spring constants enabled a more effective comparison of experimental data with theory in comparison with previously published data.

Cytochalasin B Modulates Nanomechanical Patterning and Fate in Human Adipose-Derived Stem Cells.

Cytoskeletal proteins provide architectural and signaling cues within cells. They are able to reorganize themselves in response to mechanical forces, converting the stimuli received into specific cellular responses. Thus, the cytoskeleton influences cell shape, proliferation, and even differentiation. In particular, the cytoskeleton affects the fate of mesenchymal stem cells (MSCs), which are highly attractive candidates for cell therapy approaches due to their capacity for self-renewal and multi-lineage differentiation. Cytochalasin B (CB), a cyto-permeable mycotoxin, is able to inhibit the formation of actin microfilaments, resulting in direct effects on cell biological properties. Here, we investigated for the first time the effects of different concentrations of CB (0.1-10 µM) on human adipose-derived stem cells (hASCs) both after 24 h (h) of CB treatment and 24 h after CB wash-out. CB influenced the metabolism, proliferation, and morphology of hASCs in a dose-dependent manner, in association with progressive disorganization of actin microfilaments. Furthermore, the removal of CB highlighted the ability of cells to restore their cytoskeletal organization. Finally, atomic force microscopy (AFM) revealed that cytoskeletal changes induced by CB modulated the viscoelastic properties of hASCs, influencing their stiffness and viscosity, thereby affecting adipogenic fate.

Acute cytotoxicity of mineral fibres observed by time-lapse video microscopy.

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 µg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres.