Internalization of nano- and micro-plastics in human erythrocytes leads to oxidative stress and estrogen receptor-mediated cellular responses.

Plastic material versatility has resulted in a substantial increase in its use in several sectors of our everyday lives. Consequently, concern regarding human exposure to nano-plastics (NPs) and micro-plastics (MPs) has recently increased. It has been shown that plastic particles entering the bloodstream may adhere to the erythrocyte surface and exert adverse effects following erythrocyte aggregation and adhesion to blood vessels. Here, we explored the effects of polystyrene nano-plastics (PS-NPs) and micro-plastics (PS-MPs) on human erythrocytes. Cellular morphology, binding/internalization of PS-NPs and PS-MPs, oxidative stress parameters, as well as the distribution and anion exchange capability of band 3 (anion exchanger 1; SLC4A1) have been analyzed in human erythrocytes exposed to 1 µg/mL PS-NPs or PS-MPs for 3 and 24 hours, respectively. The data obtained showed significant modifications of the cellular shape after exposure to PS-NPs or PS-MPs. In particular, a significantly increased number of acanthocytes, echinocytes and leptocytes were detected. However, the percentage of eryptotic cells (<1%) was comparable to physiological conditions. Analytical cytology and confocal microscopy showed that PS-NPs and PS-MPs bound to the erythrocyte plasma membrane, co-localized with estrogen receptors (Erα/ERß), and were internalized. An increased trafficking from the cytosol to the erythrocyte plasma membrane and abnormal distribution of ERs were also observed, consistent with ERα-mediated binding and internalization of PS-NPs. An increased phosphorylation of ERK1/2 and AKT kinases indicated that an activation of the ER-modulated non-genomic pathway occurred following exposure to PS-NPs and PS-MPs. Interestingly, PS-NPs or PS-MPs caused a significant production of reactive oxygen species, resulting in an increased lipid peroxidation and protein sulfhydryl group oxidation. Oxidative stress was also associated with an altered band 3 ion transport activity and increased oxidized haemoglobin, which led to abnormal clustering of band 3 on the plasma membrane. Taken together, these findings identify cellular events following the internalization of PS-NPs or PS-MPs in human erythrocytes and contribute to elucidating potential oxidative stress-related harmful effects, which may affect erythrocyte and systemic homeostasis.

Anti-Biofilm Activity of Oleacein and Oleocanthal from Extra-Virgin Olive Oil toward Pseudomonas aeruginosa.

New antimicrobial molecules effective against Pseudomonas aeruginosa, known as an antibiotic-resistant "high-priority pathogen", are urgently required because of its ability to develop biofilms related to healthcare-acquired infections. In this study, for the first time, the anti-biofilm and anti-virulence activities of a polyphenolic extract of extra-virgin olive oil as well as purified oleocanthal and oleacein, toward P. aeruginosa clinical isolates were investigated. The main result of our study was the anti-virulence activity of the mixture of oleacein and oleocanthal toward multidrug-resistant and intermediately resistant strains of P. aeruginosa isolated from patients with ventilator-associated pneumonia or surgical site infection. Specifically, the mixture of oleacein (2.5 mM)/oleocanthal (2.5 mM) significantly inhibited biofilm formation, alginate and pyocyanin production, and motility in both P. aeruginosa strains (p < 0.05); scanning electron microscopy analysis further evidenced its ability to inhibit bacterial cell adhesion as well as the production of the extracellular matrix. In conclusion, our results suggest the potential application of the oleacein/oleocanthal mixture in the management of healthcare-associated P. aeruginosa infections, particularly in the era of increasing antimicrobial resistance.

AAPH-induced oxidative damage reduced anion exchanger 1 (SLC4A1/AE1) activity in human red blood cells: protective effect of an anthocyanin-rich extract.

Introduction: During their lifespan in the bloodstream, red blood cells (RBCs) are exposed to multiple stressors, including increased oxidative stress, which can affect their morphology and function, thereby contributing to disease. Aim: This investigation aimed to explore the cellular and molecular mechanisms related to oxidative stress underlying anion exchanger 1 activity (band 3, SLC4A1/AE1) in human RBCs. To achieve this aim, the relationship between RBC morphology and functional and metabolic activity has been explored. Moreover, the potential protective effect of an anthocyanin-enriched fraction extracted from Callistemon citrinus flowers was studied. Methods: Cellular morphology, parameters of oxidative stress, as well as the anion exchange capability of band 3 have been analyzed in RBCs treated for 1 h with 50 mM of the pro-oxidant 2,2'-azobis (2-methylpropionamide)-dihydrochloride (AAPH). Before or after the oxidative insult, subsets of cells were exposed to 0.01 µg/mL of an anthocyanin-enriched fraction for 1 h. Results: Exposure to AAPH caused oxidative stress, exhaustion of reduced glutathione, and over-activation of the endogenous antioxidant machinery, resulting in morphological alterations of RBCs, specifically the formation of acanthocytes, increased lipid peroxidation and oxidation of proteins, as well as abnormal distribution and hyper-phosphorylation of band 3. Expected, oxidative stress was also associated with a decreased band 3 ion transport activity and an increase of oxidized haemoglobin, which led to abnormal clustering of band 3. Exposure of cells to the anthocyanin-enriched fraction prior to, but not after, oxidative stress efficiently counteracted oxidative stress-related alterations. Importantly, protection of band3 function from oxidative stress could only be achieved in intact cells and not in RBC ghosts. Conclusion: These findings contribute a) to clarify oxidative stress-related physiological and biochemical alterations in human RBCs, b) propose anthocyanins as natural antioxidants to neutralize oxidative stress-related modifications, and 3) suggest that cell integrity, and therefore a cytosolic component, is required to reverse oxidative stress-related pathophysiological derangements in human mature RBCs.

Interferon-ε as potential inhibitor of Chlamydia trachomatis infection.

Chlamydia trachomatis, the main cause of bacterial sexually transmitted diseases, is responsible for severe reproductive sequelae. Amongst all the cytokines involved in host immunity towards this pathogen, IFN-ε has recently acquired importance for its potential contribution to the female reproductive tract innate defenses. Herein, our study aimed to explore, for the first time, the activity of IFN-ε toward C. trachomatis in an in vitro infection model, by testing its effects on the different phases of chlamydial developmental cycle, as well as on the ultrastructural characteristics of chlamydial inclusions, via transmission electron microscopy. Main result is the capability of IFN-ε to alter C. trachomatis growth, as suggested by reduced infectious progenies, as well as a patchy distribution of bacteria and altered morphology of reticulate bodies within inclusions. In conclusion, our results suggest that IFN-ε could play a role in the innate and adaptive immune defenses against C. trachomatis; in the future, it will be needed to investigate its activity on an infection model more closely resembling the physiological environment of the female genital tract.

Extra Virgin Olive Oil-Based Formulations: A "Green" Strategy against Chlamydia trachomatis.

In recent decades, antibiotic misuse has emerged as an important risk factor for the appearance of multi-drug-resistant bacteria, and, recently, antimicrobial resistance has also been described in Chlamydia trachomatis as the leading cause of bacterial sexually transmitted diseases worldwide. Herein, we investigated, for the first time, the antibacterial activity against C. trachomatis of a polyphenolic extract of extra virgin olive oil (EVOO), alongside purified oleocanthal and oleacein, two of its main components, in natural deep eutectic solvent (NaDES), a biocompatible solvent. The anti-chlamydial activity of olive-oil polyphenols (OOPs) was tested in the different phases of chlamydial developmental cycle by using an in vitro infection model. Transmission and scanning electron microscopy analysis were performed for investigating potential alterations of adhesion and invasion, as well as morphology, of chlamydial elementary bodies (EBs) to host cells. The main result of our study is the anti-bacterial activity of OOPs towards C. trachomatis EBs down to a total polyphenol concentration of 1.7 µg/mL, as shown by a statistically significant decrease (93.53%) of the total number of chlamydial-inclusion-forming units (p < 0.0001). Transmission and scanning electron microscopy analysis supported its anti-chlamydial effect, suggesting that OOP might damage the chlamydial outer layers, impairing their structural integrity and hindering EB capability to infect the host cell. In conclusion, OOPs may represent an interesting alternative therapeutic option toward C. trachomatis, although further studies are necessary for exploring its clinical applications.

Interaction of Drug-Sensitive and -Resistant Human Melanoma Cells with HUVEC Cells: A Label-Free Cell-Based Impedance Study.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

Phytochemicals as Immunomodulatory Agents in Melanoma.

Cutaneous melanoma is an immunogenic highly heterogenic tumor characterized by poor outcomes when it is diagnosed late. Therefore, immunotherapy in combination with other anti-proliferative approaches is among the most effective weapons to control its growth and metastatic dissemination. Recently, a large amount of published reports indicate the interest of researchers and clinicians about plant secondary metabolites as potentially useful therapeutic tools due to their lower presence of side effects coupled with their high potency and efficacy. Published evidence was reported in most cases through in vitro studies but also, with a growing body of evidence, through in vivo investigations. Our aim was, therefore, to review the published studies focused on the most interesting phytochemicals whose immunomodulatory activities and/or mechanisms of actions were demonstrated and applied to melanoma models.

Efficacy of Combined Mechanical and Chemical Decontamination Treatments on Smooth and Rough Titanium Surfaces and Their Effects on Osteoconduction: An Ex Vivo Study.

PURPOSE: The aim of this ex vivo study was to assess the ability to remove oral biofilm by different combinations of mechanical and chemical treatments on smooth and rough titanium surfaces, as well as their impact on osteoconduction. MATERIALS AND METHODS: Forty-eight sandblasted acid-etched (SLA) and 48 machined titanium disks were contaminated with oral bacterial biofilm and exposed to the following treatments: (1) titanium brush (TB), (2) TB + 40% citric acid (CA), (3) TB + 5.25% sodium hypochlorite (NaOCl), (4) air polishing with glycine powder (AP), (5) AP + 40% CA, and (6) AP + 5.25% NaOCl. Residual bacteria and chemical contamination were assessed using viable bacterial count assay, scanning electron microscopy (SEM), and x-ray spectroscopy (XPS). Human primary osteoblast (hOB) adhesion and osteocalcin (OC) release were also evaluated. RESULTS: The microbiologic, SEM, and XPS analysis indicate a higher biofilm removal efficiency of combined mechanical-chemical treatments compared with exclusively mechanical approaches, especially on SLA surfaces. SEM analysis revealed significant alterations of surface microtopography on the disks treated with TB, while no changes were observed after AP treatment. OC release by hOBs was mainly decreased on disks treated with CA and NaOCl. CONCLUSION: The combination of mechanical and chemical treatments provides effective oral biofilm removal on both SLA and machined implant surfaces. NaOCl and CA may have a negative effect on osteoblasts cultured on SLA samples.

Label-free cell based impedance measurements of ZnO nanoparticles-human lung cell interaction: a comparison with MTT, NR, Trypan blue and cloning efficiency assays.

BACKGROUND: There is a huge body of literature data on ZnOnanoparticles (ZnO NPs) toxicity. However, the reported results are seen to be increasingly discrepant, and deep comprehension of the ZnO NPs behaviour in relation to the different experimental conditions is still lacking. A recent literature overview emphasizes the screening of the ZnO NPs toxicity with more than one assay, checking the experimental reproducibility also versus time, which is a key factor for the robustness of the results. In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of multiple independent assays. RESULTS: ECIS-measurements were compared with traditional cytotoxicity tests such as MTT, Neutral red, Trypan blue, and cloning efficiency assays. ECIS could follow the cell behavior continuously and noninvasively for days, so that certain long-term characteristics of cell proliferation under treatment with ZnO NPs were accessible. This was particularly important in the case of pro-mitogenic activity exerted by low-dose ZnO NPs, an effect not revealed by endpoint independent assays. This result opens new worrisome questions about the potential mitogenic activity exerted by ZnO NPs, or more generally by NPs, on transformed cells. Of importance, impedance curve trends (morphology) allowed to discriminate between different cell death mechanisms (apoptosis vs autophagy) in the absence of specific reagents, as confirmed by cell structural and functional studies by high-resolution microscopy. This could be advantageous in terms of costs and time spent. ZnO NPs-exposed A549 cells showed an unusual pattern of actin and tubulin distribution which might trigger mitotic aberrations leading to genomic instability. CONCLUSIONS: ZnO NPs toxicity can be determined not only by the intrinsic NPs characteristics, but also by the external conditions like the experimental setting, and this could account for discrepant data from different assays. ECIS has the potential to recapitulate the needs required in the evaluation of nanomaterials by contributing to the reliability of cytotoxicity tests. Moreover, it can overcome some false results and discrepancies in the results obtained by endpoint measurements. Finally, we strongly recommend the comparison of cytotoxicity tests (ECIS, MTT, Trypan Blue, Cloning efficiency) with the ultrastructural cell pathology studies.

How stereochemistry of lipid components can affect lipid organization and the route of liposome internalization into cells.

Though liposome-based drugs are in clinical use, the mechanism of cell internalization of liposomes is yet an object of controversy. The present experimental investigation, carried out on human glioblastoma cells, indicated different internalization routes for two diastereomeric liposomes. Molecular dynamics simulations of the lipid bilayers of the two formulations indicated that the different stereochemistry of a lipid component controls some parameters such as area per lipid molecule and fluidity of lipid membranes, surface potential and water organization at the lipid/water interface, all of which affect the interaction with biomolecules and cell components.

The Prevention of Implant Surface Alterations in the Treatment of Peri-Implantitis: Comparison of Three Different Mechanical and Physical Treatments.

The surgical treatment of peri-implantitis is currently based on the removal of biofilms from the implant surface by primary means of mechanical and physical treatments. However, such approaches often determine some alterations of the implant surface with detrimental effects on re-osseointegration. This study aims to evaluate the effects of four different mechanical and physical treatments on titanium samples with moderately rough surface. Air powder abrasion (AP) with glycine powder, a titanium brush (TB) and a diode laser at 3 W (L3) and 4 W (L4) were tested. Surface morphology, roughness and chemical composition were then assessed by scanning electron microscope (SEM), white light interferometer and X-ray photoelectron spectroscopy (XPS), respectively. The microscopic analysis revealed significant alterations in surface morphology on TB samples, while AP and L3 had only a minor or null impact. L4 samples revealed signs of overheating due to the excessive power. Nevertheless, the overall roughness of the samples was not significantly altered in terms of roughness parameters. Similarly, surface chemical composition was not significantly affected by the treatments. Among the treatments tested in this study, air powder abrasion with glycine powder and 3 W diode laser had the lowest impact on surface physicochemical properties.

Structurally related glucosylated liposomes: Correlation of physicochemical and biological features.

Liposomes functionalized on their surface with carbohydrates (glycoliposomes) represent an optimal approach for targeting of drugs to diseased tissues in vivo, thanks to biocompatibility, low toxicity and easy manufacturing of these lipid nanoparticles. Here we report on the study of liposomes including a novel glycosylated amphiphile and on the comparison of their features with those of glycosylated analogues described previously. Further, the capability of the different glucosylated formulations to interact with three breast cancer cell lines was investigated. Our results show that the hydrophobic portion of the lipid bilayer strongly influences both the properties and the internalization of glycosylated liposomes.

The proneural gene ASCL1 governs the transcriptional subgroup affiliation in glioblastoma stem cells by directly repressing the mesenchymal gene NDRG1.

Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.

Biomimetic Implant Surface Functionalization with Liquid L-PRF Products: In Vitro Study.

OBJECTIVE: Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products. METHODS: Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in (1) a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes, (2) an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and (3) the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM). RESULTS: Under microscopic observation, (1) the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood; (2) in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and (3) in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating. CONCLUSIONS: Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products.

The product of the human AHI-1 (Abelson helper integration site) gene: experimental in vitro data point to its involvement in tumor cell invasion.

INTRODUCTION: Each of the steps involved in invasion of tumors requires specific molecular program in which the modulation of adhesive and migratory properties of disseminating cells plays an essential role. The improvement in the knowledge of these mechanisms can lead to discovery of new target candidates in drug development. In this study we focused attention on the product of the human AHI-1 (Abelson helper integration site) gene Jouberin (Jbn). METHODS: In particular, we explore by in vitro invasion assay, AHI-1 knockdown and electron microscopy, if Jbn is involved in the signaling machinery that regulates tumor invasion. To this purpose tumor cells of different histological derivation (brain, breast, skin) were employed. RESULTS: We found that Jbn expression correlates with the proliferation, invasive potential and invasion strategy of the tested tumor cells, and that its downregulation reduces their capability of migrating and invading the extracellular matrix. CONCLUSIONS: The results obtained in this study for the first time point to Jbn as a new candidate involved in the invasion process of tumor cells, and as potential molecular target in anticancer therapy.

Phosphatidylcholine-specific phospholipase C inhibition down- regulates CXCR4 expression and interferes with proliferation, invasion and glycolysis in glioma cells.

BACKGROUND: The chemokine receptor CXCR4 plays a crucial role in tumors, including glioblastoma multiforme (GBM), the most aggressive glioma. Phosphatidylcholine-specific phospholipase C (PC-PLC), a catabolic enzyme of PC metabolism, is involved in several aspects of cancer biology and its inhibition down-modulates the expression of growth factor membrane receptors interfering with their signaling pathways. In the present work we investigated the possible interplay between CXCR4 and PC-PLC in GBM cells. METHODS: Confocal microscopy, immunoprecipitation, western blot analyses, and the evaluation of migration and invasion potential were performed on U87MG cells after PC-PLC inhibition with the xanthate D609. The intracellular metabolome was investigated by magnetic resonance spectroscopy; lactate levels and lactate dehydrogenase (LDH) activity were analyzed by colorimetric assay. RESULTS: Our studies demonstrated that CXCR4 and PC-PLC co-localize and are associated on U87MG cell membrane. D609 reduced CXCR4 expression, cell proliferation and invasion, interfering with AKT and EGFR activation and expression. Metabolic analyses showed a decrease in intracellular lactate concentration together with a decrement in LDH activity. CONCLUSIONS: Our data suggest that inhibition of PC-PLC could represent a new molecular approach in glioma biology not only for its ability in modulating cell metabolism, glioma growth and motility, but also for its inhibitory effect on crucial molecules involved in cancer progression.

Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model.

BACKGROUND: The CXCL12/CXCR4 pathway regulates tumor cell proliferation, metastasis, angiogenesis and the tumor-microenvironment cross-talk in several solid tumors, including glioblastoma (GBM), the most common and fatal brain cancer. In the present study, we evaluated the effects of peptide R, a new specific CXCR4 antagonist that we recently developed by a ligand-based approach, in an in vitro and in vivo model of GBM. The well-characterized CXCR4 antagonist Plerixafor was also included in the study. METHODS: The effects of peptide R on CXCR4 expression, cell survival and migration were assessed on the human glioblastoma cell line U87MG exposed to CXCL12, by immunofluorescence and western blotting, MTT assay, flow cytometry and transwell chamber migration assay. Peptide R was then tested in vivo, by using U87MG intracranial xenografts in CD1 nude mice. Peptide R was administered for 23 days since cell implantation and tumor volume was assessed by magnetic resonance imaging (MRI) at 4.7 T. Glioma associated microglia/macrophage (GAMs) polarization (anti-tumor M1 versus pro-tumor M2 phenotypes) and expressions of vascular endothelial growth factor (VEGF) and CD31 were assessed by immunohistochemistry and immunofluorescence. RESULTS: We found that peptide R impairs the metabolic activity and cell proliferation of human U87MG cells and stably reduces CXCR4 expression and cell migration in response to CXCL12 in vitro. In the orthotopic U87MG model, peptide R reduced tumor cellularity, promoted M1 features of GAMs and astrogliosis, and hindered intra-tumor vasculature. CONCLUSIONS: Our findings suggest that targeting CXCR4 by peptide R might represent a novel therapeutic approach against GBM, and contribute to the rationale to further explore in more complex pre-clinical settings the therapeutic potential of peptide R, alone or in combination with standard therapies of GBM.

Migratory behaviour of tumour cells: a scanning electron microscopy study.

BACKGROUND: Tumour cells utilize different migration strategies to invade surrounding tissues and elude anticancer treatments. It is therefore important to understand the mechanisms underlying migration process, in order to aid the development of therapies aimed at blocking the dissemination of cancer cells. AIMS: In this study tumour cell lines of different histological origin were analysed by combining 2D and 3D in vitro assays, biochemical tests and high resolution imaging by scanning electron microscopy (SEM) in order to look insight strategies adopted by tumour cells to invade extracellular matrix. RESULTS: Quantitative (computer-assisted colour camera equipped-light microscopy) and qualitative analysis (SEM) indicated that the most aggressive tumour cells adopt an "individual" behaviour. The analysis of intracellular signalling demonstrated that the highest invasive potential was associated with the activation of AKT, ERK, FAK and ERM proteins. The "individual" behaviour was positively related to the expression of VLA-2 and inversely related with the E-cadherin expression. CONCLUSIONS: The combination of 2D and 3D in vitro assays, biochemical tests and ultrastructural investigations proved to be a suitable test for the investigation of tumour cell migration and invasion. The high resolution imaging by SEM highlighted the interrelationships between cells in different migratory behaviours of tumour cells.

Liposomes as nanomedical devices.

Since their discovery in the 1960s, liposomes have been studied in depth, and they continue to constitute a field of intense research. Liposomes are valued for their biological and technological advantages, and are considered to be the most successful drug-carrier system known to date. Notable progress has been made, and several biomedical applications of liposomes are either in clinical trials, are about to be put on the market, or have already been approved for public use. In this review, we briefly analyze how the efficacy of liposomes depends on the nature of their components and their size, surface charge, and lipidic organization. Moreover, we discuss the influence of the physicochemical properties of liposomes on their interaction with cells, half-life, ability to enter tissues, and final fate in vivo. Finally, we describe some strategies developed to overcome limitations of the "first-generation" liposomes, and liposome-based drugs on the market and in clinical trials.

Brain tumor stem cell dancing.

BACKGROUND: Issues regarding cancer stem cell (CSC) movement are important in neurosphere biology as cell-cell or cell-environment interactions may have significant impacts on CSC differentiation and contribute to the heterogeneity of the neurosphere. AIMS: Despite the growing body of literature data on the biology of brain tumor stem cells, floating CSC-derived neurospheres have been scarcely characterized from a morphological and ultrastructural point of view. RESULTS: Here we report a morphological and ultrastructural characterization performed by live imaging and scanning electron microscopy. Glioblastoma multiforme (GBM) CSC-derived neurospheres are heterogeneous and are constituted by cells, morphologically different, capable of forming highly dynamic structures. These dynamic structures are regulated by not serendipitous cell-cell interactions, and they synchronously pulsate following a cyclic course made of "fast" and "slow" alternate phases. Autocrine/paracrine non canonical Wnt signalling appears to be correlated with the association status of neurospheres. CONCLUSIONS: The results obtained suggest that GBM CSCs can behave both as independents cells and as "social" cells, highly interactive with other members of its species, giving rise to a sort of "multicellular organism".