ABSTRACT
Extracellular vesicles (EVs) enriched with bioactive molecules have gained considerable attention in nanotechnology because they are critical to intercellular communication while maintaining low immunological impact. Among biological matrices, urine has emerged as a noninvasive source of extracellular-contained liquid biopsy, currently of interest as a readout for physiological adaptations. Therefore, we aimed to evaluate chronic adaptations of endurance sport practice in terms of urinary EV parameters and evaluated by food consumption assessment. Two balanced groups of 13 inactive controls vs. triathlon athletes were enrolled; their urinary EVs were obtained by differential ultracentrifugation and analyzed by dynamic light scattering and transmission electron and atomic force microscopy. The cargo was analyzed by means of purine and miRNA content through HPLC-UV and qRT-PCR. Specific urinary EV signatures differentiated inactive versus endurance-trained in terms of peculiar shape. Particularly, a spheroid shape, smaller size, and lower roughness characterize EVs from triathletes. Metabolic and regulatory miRNAs often associated with skeletal muscle (i.e., miR378a-5p, miR27a-3p, miR133a, and miR206) also accounted for a differential signature. These miRNAs and guanosine in urinary EVs can be used as a readout for metabolic status along with the shape and roughness of EVs, novel informative parameters that are rarely considered. The network models allow scholars to entangle nutritional and exercise factors related to EVs' miRNA and purine content to depict metabolic signatures. All in all, multiplex biophysical and molecular analyses of urinary EVs may serve as promising prospects for research in exercise physiology.
Subject(s)
Body Fluids , Extracellular Vesicles , MicroRNAs , Urinary Tract , Humans , MicroRNAs/metabolism , Urinary Tract/metabolism , Extracellular Vesicles/metabolism , Body Fluids/metabolism , Purines/metabolismABSTRACT
Cellulose acetate (CA) nanoparticles were combined with two antimicrobial agents, namely lemongrass (LG) essential oil and Cu-ferrite nanoparticles. The preparation method of CA nanocapsules (NCs), with the two antimicrobial agents, was based on the nanoprecipitation method using the solvent/anti-solvent technique. Several physical and chemical analyses were performed to characterize the resulting NCs and to study their formation mechanism. The size of the combined antimicrobial NCs was found to be ca. 220 nm. The presence of Cu-ferrites enhanced the attachment of LG essential oil into the CA matrix. The magnetic properties of the combined construct were weak, due to the shielding of Cu-ferrites from the polymeric matrix, making them available for drug delivery applications where spontaneous magnetization effects should be avoided. The antimicrobial properties of the NCs were significantly enhanced with respect to CA/LG only. This work opens novel routes for the development of organic/inorganic nanoparticles with exceptional antimicrobial activities.
Subject(s)
Cellulose/analogs & derivatives , Copper/pharmacology , Cymbopogon/chemistry , Ferric Compounds/chemistry , Oils, Volatile/pharmacology , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Cellulose/chemistry , Copper/chemistry , Microbial Sensitivity Tests , Nanocapsules/chemistry , Oils, Volatile/chemistry , Particle Size , Staphylococcus aureus/drug effectsABSTRACT
Toxicity of silver nanoparticles (AgNPs) is supported by many observations in literature, but no mechanism details have been proved yet. Here we confirm and quantify the toxic potential of fully characterized AgNPs in HeLa and A549 cells. Notably, through a specific fluorescent probe, we demonstrate the intracellular release of Ag(+) ions in living cells after nanoparticle internalization, showing that in-situ particle degradation is promoted by the acidic lysosomal environment. The activation of metallothioneins in response to AgNPs and the possibility to reverse the main toxic pathway by Ag(+) chelating agents demonstrate a cause/effect relationship between ions and cell death. We propose that endocytosed AgNPs are degraded in the lysosomes and the release of Ag(+) ions in the cytosol induces cell damages, while ions released in the cell culture medium play a negligible effect. These findings will be useful to develop safer-by-design nanoparticles and proper regulatory guidelines of AgNPs. From the clinical editor: The authors describe the toxic potential of silver nanoparticles (AgNP) in human cancer cell lines. Cell death following the application of AgNPs is dose-dependent, and it is mostly due to Ag+ ions. Further in vivo studies should be performed to gain a comprehensive picture of AgNP-toxicity in mammals.
Subject(s)
Cytosol/metabolism , Metal Nanoparticles/chemistry , Silver , Cations, Monovalent/pharmacokinetics , HeLa Cells , Humans , Lysosomes/metabolism , Silver/chemistry , Silver/pharmacokinetics , Silver/pharmacologyABSTRACT
DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.
Subject(s)
Chemistry Techniques, Analytical/methods , DNA/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , MicroRNAs/analysis , Biological Assay , DNA/metabolism , Fluorescence , Limit of Detection , MicroRNAs/chemistry , Molecular Probes/chemistryABSTRACT
A facile green chemistry approach for the synthesis of sub-5 nm silver and gold nanoparticles is reported. The synthesis was achieved by a photochemical method using tyrosine as the photoreducing agent. The size of the gold and silver nanoparticles was about 3 and 4 nm, respectively. The nanoparticles were characterized using x-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy and photoluminescence spectroscopy. Both silver and gold nanoparticles synthesized by this method exhibited fluorescence properties and their use for cell imaging applications has been demonstrated.
Subject(s)
Fluorescent Dyes/chemistry , Metal Nanoparticles/chemistry , Nanotechnology/methods , Photochemistry/methods , Cell Line, Tumor , Gold/chemistry , Green Chemistry Technology , HeLa Cells , Humans , Metals/chemistry , Microscopy, Confocal , Microscopy, Electron, Transmission , Oxygen/chemistry , Silver/chemistry , Solvents/chemistry , Spectrophotometry , Spectroscopy, Fourier Transform Infrared , Tetrazolium Salts/chemistry , Water/chemistry , X-Ray DiffractionABSTRACT
Wound dressing production represents an important segment in the biomedical healthcare field, but finding a simple and eco-friendly method that combines a natural compound and a biocompatible dressing production for biomedical application is still a challenge. Therefore, the aim of this study is to develop wound healing dressings that are environmentally friendly, low cost, and easily produced, using natural agents and a physical crosslinking technique. Hydrogel wound healing dressings were prepared from polyvinyl alcohol/carboxymethyl cellulose and sericin using the freeze-thawing method as a crosslinking method. The morphological characterization was carried out by scanning electron microscopy (SEM), whereas the mechanical analysis was carried out by dynamic mechanical analysis (DMA) to test the tensile strength and compression properties. Then, the healing property of the wound dressing material was tested by in vitro and ex vivo tests. The results show a three-dimensional microporous structure with no cytotoxicity, excellent stretchability with compressive properties similar to those of human skin, and excellent healing properties. The proposed hydrogel dressing was tested in vitro with HaCaT keratinocytes and ex vivo with epidermal tissues, demonstrating an effective advantage on wound healing acceleration. Accordingly, this study was successful in developing wound healing dressings using natural agents and a simple and green crosslinking method.
ABSTRACT
The peculiar physical/chemical characteristics of engineered nanomaterials have led to a rapid increase of nanotechnology-based applications in many fields. However, before exploiting their huge and wide potential, it is necessary to assess their effects upon interaction with living systems. In this context, the screening of nanomaterials to evaluate their possible toxicity and understand the underlying mechanisms currently represents a crucial opportunity to prevent severe harmful effects in the next future. In this work we show the in vivo toxicity of gold nanoparticles (Au NPs) in Drosophila melanogaster, highlighting significant genotoxic effects and, thus, revealing an unsettling aspect of the long-term outcome of the exposure to this nanomaterial. After the treatment with Au NPs, we observed dramatic phenotypic modifications in the subsequent generations of Drosophila, demonstrating their capability to induce mutagenic effects that may be transmitted to the descendants. Noteworthy, we were able to obtain the first nanomaterial-mutated organism, named NM-mut. Although these results sound alarming, they underline the importance of systematic and reliable toxicology characterizations of nanomaterials and the necessity of significant efforts by the nanoscience community in designing and testing suitable nanoscale surface engineering/coating to develop biocompatible nanomaterials with no hazardous effects for human health and environment. FROM THE CLINICAL EDITOR: While the clinical application of nanomedicine is still in its infancy, the rapid evolution of this field will undoubtedly result in a growing number of clinical trials and eventually in human applications. The interactions of nanoparticles with living organisms determine their toxicity and long-term safety, which must be properly understood prior to large-scale applications are considered. The paper by Dr. Pompa's team is the first ever demonstration of mutagenesis resulting in clearly observable phenotypic alterations and the generation of nano-mutants as a result of exposure to citrate-surfaced gold nanoparticles in drosophila. These groundbreaking results are alarming, but represent a true milestone in nanomedicine and serve as a a reminder and warning about the critical importance of "safety first" in biomedical science.
Subject(s)
Drosophila melanogaster/genetics , Gold/adverse effects , Metal Nanoparticles/adverse effects , Mutagenesis/genetics , Animals , Drosophila Proteins/genetics , Gene Expression , Gold/chemistry , Hemocytes/cytology , Humans , In Situ Nick-End Labeling , Metal Nanoparticles/chemistry , Mutagenicity Tests , Phenotype , Safety , Tumor Suppressor Protein p53/geneticsABSTRACT
Metabolic disorders such as the highly prevalent disease diabetes require constant monitoring. The health status of patients is linked to glucose levels in blood, which are typically measured invasively, but can also be correlated to other body fluids such as sweat. Aiming at a reliable glucose biosensor, an enzymatic sensing layer was fabricated on flexible polystyrene foil, for which a versatile nanoimprinting process for microfluidics was presented. For the sensing layer, a gold electrode was modified with a cysteine layer and glutaraldehyde cross-linker for enzyme conformal immobilization. Chronoamperometric measurements were conducted in PBS buffered glucose solution at two potentials (0.65 V and 0.7 V) and demonstrated a linear range between 0.025 mM to 2mM and an operational range of 0.025 mM to 25 mM. The sensitivity was calculated as 1.76µA/mM/cm2 and the limit of detection (LOD) was calculated as 0.055 mM at 0.7 V. An apparent Michaelis-Menten constant of 3.34 mM (0.7 V) and 0.445 mM (0.65 V) was computed. The wide operational range allows the application for point-of-care testing for a variety of body fluids. Yet, the linear range and low LOD make this biosensor especially suitable for non-invasive sweat sensing wearables.
ABSTRACT
Titanium dioxide nanoparticles (TiO2NPs), in the two crystalline forms, rutile and anatase, have been widely used in many industrial fields, especially in cosmetics. Therefore, a lot of details about their safety issues have been discussed by the scientific community. Many studies have led to a general agreement about TiO2NPs toxicity, in particular for anatase form, but no mechanism details have been proved yet. In this study, data confirm the different toxic potential of rutile and anatase TiO2NPs in two cell lines up to 5nM nanoparticles concentration. Moreover, we evaluated the role of titanium ions released by TiO2NPs in different conditions, at pH=4.5 (the typical lysosomal compartment pH) and at pH=5.5 (the skin physiological pH) in conditions of darkness and light, to mimic the dermal exposure of cosmetics. Anatase nanoparticles were proner to degradation both in the acidic conditions and at skin pH. Our study demonstrates that pH and sunlight are dominant factors to induce oxidative stress, TiO2NPs degradation and toxicity effects.
Subject(s)
Metal Nanoparticles/toxicity , Titanium/toxicity , A549 Cells , Caspase 3/metabolism , Humans , Hydrogen-Ion Concentration , L-Lactate Dehydrogenase/metabolism , MCF-7 Cells , Membrane Potential, Mitochondrial/drug effects , Metal Nanoparticles/chemistry , Metal Nanoparticles/radiation effects , Oxidative Stress , Sunlight , Titanium/chemistry , Titanium/radiation effectsABSTRACT
One of the current challenges in wound care is the development of multifunctional dressings that can both protect the wound from external agents and promote the regeneration of the new tissue. Here, we show the combined use of two naturally derived compounds, sodium alginate and lavender essential oil, for the production of bioactive nanofibrous dressings by electrospinning, and their efficacy for the treatment of skin burns induced by midrange ultraviolet radiation (UVB). We demonstrate that the engineered dressings reduce the risk of microbial infection of the burn, since they stop the growth of Staphylococcus aureus. Furthermore, they are able to control and reduce the inflammatory response that is induced in human foreskin fibroblasts by lipopolysaccharides, and in rodents by UVB exposure. In particular, we report a remarkable reduction of pro-inflammatory cytokines when fibroblasts or animals are treated with the alginate-based nanofibers. The down-regulation of cytokines production and the absence of erythema on the skin of the treated animals confirm that the here described dressings are promising as advanced biomedical devices for burn management.
ABSTRACT
Nanocapsules and nanoparticles play an essential role in the delivery of pharmaceutical agents in modern era, since they can be delivered in specific tissues and cells. Natural polymers, such as cellulose acetate, are becoming very important due to their availability, biocompatibility, absence of toxicity and biodegradability. In parallel, essential oils are having continuous growth in biomedical applications due to the inherent active compounds that they contain. A characteristic example is lemongrass oil that has exceptional antimicrobial properties. In this work, nanocapsules of cellulose acetate with lemongrass oil were developed with the solvent/anti-solvent method with resulting diameter tailored between 95 and 185nm. Various physico-chemical and surface analysis techniques were employed to investigate the formation of the nanocapsules. These all-natural nanocapsules found to well bioadhere to mucous membranes and to have very good antimicrobial properties at little concentrations against Escherichia coli and Staphylococcus aureus.
Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Cellulose/analogs & derivatives , Nanocapsules/chemistry , Oils, Volatile/chemistry , Plant Oils/chemistry , Plant Oils/pharmacology , Terpenes/chemistry , Terpenes/pharmacology , Cellulose/chemistry , Escherichia coli/drug effects , Microbial Sensitivity Tests/methods , Oils, Volatile/pharmacology , Solvents/chemistry , Staphylococcus aureus/drug effectsABSTRACT
Cancer cell motility is one of the major events involved in metastatic process. Tumor cells that disseminate from a primary tumor can migrate into the vascular system and, being carried by the bloodstream, transmigrate across the endothelium, giving rise to a new tumor site. However, during the invasive process, tumor cells must pass through the extracellular matrix, whose structural and mechanical properties define the parameters of the migration process. Here, we propose 3D-complex cage-like microstructures, realized by two-photon (TP) direct laser writing (DLW), to analyze cell migration through pores significantly smaller than the cell nucleus. We found that the ability to traverse differently sized pores depends on the metastatic potential and on the invasiveness of the cell lines, allowing to establish a pore-area threshold value able to discriminate between non-tumorigenic and tumorigenic human breast cells.
Subject(s)
Microscopy, Confocal , Time-Lapse Imaging , Cell Line, Tumor , Cell Movement , Coculture Techniques , Humans , Nonmuscle Myosin Type IIA/metabolismABSTRACT
We have studied in vitro toxicity of iron oxide nanoparticles (NPs) coated with a thin silica shell (Fe3O4/SiO2 NPs) on A549 and HeLa cells. We compared bare and surface passivated Fe3O4/SiO2 NPs to evaluate the effects of the coating on the particle stability and toxicity. NPs cytotoxicity was investigated by cell viability, membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assays, and their genotoxicity by comet assay. Our results show that NPs surface passivation reduces the oxidative stress and alteration of iron homeostasis and, consequently, the overall toxicity, despite bare and passivated NPs show similar cell internalization efficiency. We found that the higher toxicity of bare NPs is due to their stronger in-situ degradation, with larger intracellular release of iron ions, as compared to surface passivated NPs. Our results indicate that surface engineering of Fe3O4/SiO2 NPs plays a key role in improving particles stability in biological environments reducing both cytotoxic and genotoxic effects.
Subject(s)
Coated Materials, Biocompatible/toxicity , Ferric Compounds/toxicity , Materials Testing , Nanoparticles/toxicity , Nanotechnology/methods , Silicon Dioxide/toxicity , Toxicity Tests , Cell Survival/drug effects , Culture Media/pharmacology , DNA Damage , Endocytosis/drug effects , HeLa Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Nanoparticles/ultrastructure , Reactive Oxygen Species/metabolism , Surface PropertiesABSTRACT
The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment--where particles are abundantly internalized--is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments.
Subject(s)
Apoptosis/drug effects , Cell Membrane/chemistry , Endocytosis/physiology , Gold/toxicity , Metal Nanoparticles/toxicity , Diffusion , Dose-Response Relationship, Drug , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Particle SizeABSTRACT
We show that water soluble InP/ZnS core/shell QDs are a safer alternative to CdSe/ZnS QDs for biological applications, by comparing their toxicity in vitro (cell culture) and in vivo (animal model Drosophila). By choosing QDs with comparable physical and chemical properties, we find that cellular uptake and localization are practically identical for these two nanomaterials. Toxicity of CdSe/ZnS QDs appears to be related to the release of poisonous Cd(2+) ions and indeed we show that there is leaching of Cd(2+) ions from the particle core despite the two-layer ZnS shell. Since an almost identical amount of In(III) ions is observed to leach from the core of InP/ZnS QDs, their very low toxicity as revealed in this study hints at a much lower intrinsic toxicity of indium compared to cadmium.
Subject(s)
Cadmium Compounds/toxicity , Drosophila/drug effects , Indium/toxicity , Phosphines/toxicity , Selenium Compounds/toxicity , Zinc Compounds/toxicity , Animals , Materials Testing , Quantum Dots , Survival RateABSTRACT
Bacterial adhesion onto abiotic surfaces is an important issue in biology and medicine since understanding the bases of such interaction represents a crucial aspect in the design of safe implant devices with intrinsic antibacterial characteristics. In this framework, we investigated the effects of nanostructured metal substrates on Escherichia coli adhesion and adaptation in order to understand the bio-molecular dynamics ruling the interactions at the interface. In particular, we show how highly controlled nanostructured gold substrates impact the bacterial behavior in terms of morphological changes and lead to modifications in the expression profile of several genes, which are crucially involved in the stress response and fimbrial synthesis. These results mainly demonstrate that E. coli cells are able to sense even slight changes in surface nanotopography and to actively respond by activating stress-related pathways. At the same time, our findings highlight the possibility of designing nanoengineered substrates able to trigger specific bio-molecular effects, thus opening the perspective of smartly tuning bacterial behavior by biomaterial design.
ABSTRACT
Despite the extensive use of silica nanoparticles (SiO(2)NPs) in many fields, the results about their potential toxicity are still controversial. In this work, we have performed a systematic in vitro study to assess the biological impact of SiO(2)NPs, by investigating 3 different sizes (25, 60 and 115 nm) and 2 surface charges (positive and negative) of the nanoparticles in 5 cell lines (3 in adherence and 2 in suspension). We analyzed the cellular uptake and distribution of the NPs along with their possible effects on cell viability, membrane integrity and generation of reactive oxygen species (ROS). Experimental results show that all the investigated SiO(2)NPs do not induce detectable cytotoxic effects (up to 2.5 nM concentration) in all cell lines, and that cellular uptake is mediated by an endocytic process strongly dependent on the particle size and independent of its original surface charge, due to protein corona effects. Once having assessed the biocompatibility of SiO(2)NPs, we have evaluated their potential in gene delivery, showing their ability to silence specific protein expression. The results of this work indicate that monodisperse and stable SiO(2)NPs are not toxic, revealing their promising potential in various biomedical applications.
Subject(s)
Cell Survival/drug effects , DNA/administration & dosage , DNA/genetics , Gene Silencing , Nanocapsules/chemistry , Nanocapsules/toxicity , Silicon Dioxide/toxicity , Transfection/methods , Cell Line , HumansABSTRACT
The expected potential benefits promised by nanotechnology in various fields have led to a rapid increase of the presence of engineered nanomaterials in a high number of commercial goods. This is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of the physical/chemical factors responsible for their toxic effects. In this work, we evaluated the toxicity of monodisperse citrate-capped gold nanoparticles (AuNPs) of different sizes (5, 15, 40, and 80 nm) in the model organism Drosophila melanogaster, upon ingestion. To properly evaluate and distinguish the possible dose- and/or size-dependent toxicity of the AuNPs, we performed a thorough assessment of their biological effects, using two different dose-metrics. In the first approach, we kept constant the total surface area of the differently sized AuNPs (Total Exposed Surface area approach, TES), while, in the second approach, we used the same number concentration of the four different sizes of AuNPs (Total Number of Nanoparticles approach, TNN). We observed a significant AuNPs-induced toxicity in vivo, namely a strong reduction of Drosophila lifespan and fertility performance, presence of DNA fragmentation, as well as a significant modification in the expression levels of genes involved in stress responses, DNA damage recognition and apoptosis pathway. Interestingly, we found that, within the investigated experimental conditions, the toxic effects in the exposed organisms were directly related to the concentration of the AuNPs administered, irrespective of their size.
Subject(s)
Body Size/physiology , Citric Acid/toxicity , Drosophila melanogaster/drug effects , Gold/chemistry , Metal Nanoparticles/toxicity , Animals , Body Size/drug effects , Citric Acid/chemistry , Citric Acid/pharmacology , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/metabolism , Drosophila melanogaster/physiology , Environmental Exposure , Female , Fertility/drug effects , Fertility/physiology , Gold/pharmacology , Gold/toxicity , In Situ Nick-End Labeling , Longevity/drug effects , Longevity/physiology , Male , Metal Nanoparticles/chemistry , Osmolar Concentration , Reactive Oxygen Species/metabolism , Reproduction/drug effects , Reproduction/physiologyABSTRACT
Bacterial adhesion onto inorganic/nanoengineered surfaces is a key issue in biotechnology and medicine, because it is one of the first necessary steps to determine a general pathogenic event. Understanding the molecular mechanisms of bacteria-surface interaction represents a milestone for planning a new generation of devices with unanimously certified antibacterial characteristics. Here, we show how highly controlled nanostructured substrates impact the bacterial behavior in terms of morphological, genomic, and proteomic response. We observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM) that type-1 fimbriae typically disappear in Escherichia coli adherent onto nanostructured substrates, as opposed to bacteria onto reference glass or flat gold surfaces. A genetic variation of the fimbrial operon regulation was consistently identified by real time qPCR in bacteria interacting with the nanorough substrates. To gain a deeper insight into the molecular basis of the interaction mechanisms, we explored the entire proteomic profile of E. coli by 2D-DIGE, finding significant changes in the bacteria adherent onto the nanorough substrates, such as regulations of proteins involved in stress processes and defense mechanisms. We thus demonstrated that a pure physical stimulus, that is, a nanoscale variation of surface topography, may play per se a significant role in determining the morphological, genetic, and proteomic profile of bacteria. These data suggest that in depth investigations of the molecular processes of microorganisms adhering to surfaces are of great importance for the design of innovative biomaterials with active biological functionalities.
Subject(s)
Bacterial Adhesion/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Nanostructures/chemistry , Nanostructures/ultrastructure , Escherichia coli/classification , Escherichia coli/cytology , Genomics , Macromolecular Substances/chemistry , Materials Testing , Molecular Conformation , Particle Size , Species Specificity , Surface PropertiesABSTRACT
BACKGROUND AND AIM: The mechanisms of hepatocarcinogenesis induced by hepatitis C virus remain unclear. Our aim was to investigate the effect of the HCV core protein on the promoter methylation status of selected genes potentially involved in the hepatocellular carcinoma (HCC). MATERIALS AND METHODS: We evaluated the promoter methylation levels of the E-cadherin (CDH1), the glutathione S-transferase p1 (GSTP1), adenomatosis polyposis coli (APC), tissue inhibitor of metalloproteinase 3 (TIMP3), catenin (cadherin-associated protein) beta 1 (CNNTB1) genes by a quantitative methylation-specific polymerase chain reaction (QMSP) in the in vitro model of Huh-7 cells expressing the HCV core protein of genotype 1b. RESULTS: We found that CDH1 promoter was hypermethylated in genotype 1b HCV core protein-positive cells as compared to control cells expressing the GFP protein alone (HCV core 1b vs GFP p=0.00; HCV core 1b vs Huh-7 p=0.03). This resulted in reduced levels of CDH1 protein as evaluated by immunoblot and by immunofluorescence. On the other hand no significant changes were observed for the other genes investigated. Furthermore, we present evidence that genotype 1b HCV core protein expression induces SIRT1 upregulation and that treatment with SIRT1 inhibitor sirtinol decreases the methylation levels of CDH1 promoter (1b+sirtinol vs 1b p=0.05; 1b+sirtinol vs GFP+sirtinol p=NS) resulting in 1.7-fold increased CDH1 mRNA expression (1b+sirtinol vs 1b p=0.05). CONCLUSIONS: Our findings suggest that HCV core protein could play a role in HCC at least in part by altering the methylation status of CDH1 promoter. These findings could also suggest a novel therapeutic approach for HCC.