Conventional approaches to human intracellular optical sensing, generally, require dedicated laboratories with bulky detection systems. They are performed by cell labeling procedures based on the use ...of fluorophores that are, mostly, phototoxic, invasive, bleached in case of prolonged light exposures, which require carriers and/or structural modifications for the cellular uptake. These issues, together with the sensitivity of the eukaryotic cell model, could be problematic towards the development of a robust sensing system suitable for biomedical screening. In this work, we studied a sensing system resulting from the combination of the commercial tris(2,2’bipyridyl)ruthenium(II) fluorophore, for cell labeling, with a potentially miniaturizable optical system composed by a laser source and a photomultiplier tube, for the fluorescence analysis.
Cytokines belonging to the TNF superfamily play a relevant role in neurodegenerative processes. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), released during neuronal injury, has ...proven to potently mediate and sustain neurotoxic processes leading to neuronal death. Similarly to TRAIL, the cytokine Glucocorticoid-induced TNF receptor ligand (GITRL) is able to transduce proapoptotic signals. In spite of the array of reports suggesting relationships between TRAIL and other cytokines, scanty data are, so far, available about a GITRL/TRAIL crosstalk.
Here, we investigated possible interactions between TRAIL and the GITRL system in an in vitro model of neurodegeneration, using the human cortical neuronal cell line HCN-2. Cultured HCN-2 neurons were incubated at different times with GITRL and/or TRAIL, and thereafter nucleic acid and protein expression were measured. Real-time PCR analysis showed that the human cortical neuronal cell line HCN-2 does not express GITRL mRNA, but the latter is induced after treatment with TRAIL. In addition, HCN-2 cells did not express the GITRL receptor GITR mRNA, neither in control cultures, nor after treatment with TRAIL. All mRNA data were confirmed by western blot analysis of proteins. Cell viability assay showed that TRAIL, when associated to GITRL, was able to exert additive toxic effects. A counterproof was provided in experiments performed blocking GITRL, in which TRAIL-mediated toxicity appeared significantly reduced. Results suggest that GITRL/TRAIL redundancy during neurodegenerative processes implies extended potentiation of detrimental effects of both cytokines on neurons, eventually leading to larger cell damage and death.
Finally, characterization of novel molecular targets within the TRAIL/GITRL interplay may represent a platform for innovative therapy of neurodegenerative disorders.
A compositional analysis on a set of human genes classified in several functional classes was performed. We found out that the GC3, i.e. the GC level at the third codon positions, of the genes ...involved in cellular metabolism was significantly higher than those involved in information storage and processing. Analyses of human/Xenopus ortologous genes showed that: (i) the GC3 increment of the genes involved in cellular metabolism was significantly higher than those involved in information storage and processing; and (ii) a strong correlation between the GC3 and the corresponding GCi, i.e. the GC level of introns, was found in each functional class. The non-randomness of the GC increments favours the selective hypothesis of gene/genome evolution.
The effect of the nanoparticles on the marine organisms, depends on their size, chemical composition, surface structure, solubility and shape. In order to take advantage from their activity, ...preserving the surrounding environment from a possible pollution, we are trying to trap the nanoparticles into new nanomaterials. The nanomaterials tested were synthesized proposing a ground-breaking approach by an upside-down vision of the Au/TiO 2 nano-system to avoid the release of nanoparticles. The system was synthesized by wrapping Au nanoparticles with a thin layer of TiO 2 . The non-toxicity of the nano-system was established by testing the effect of the material on zebrafish larvae. Danio rerio o zebrafish was considered an excellent model for the environmental biomonitoring of aquatic environments and the Zebrafish Embryo Toxicity Test (ZFET) is considered an alternative method of animal test. For this reason zebrafish larvae were exposed to different concentrations of nanoparticles of TiO 2 and Au and new nanomaterials. As biomarkers of exposure, we evaluated the expression of metallothioneins by immunohistochemistry analysis and western blotting analysis also. The results obtained by toxicity test showed that neither mortality as well as sublethal effects were induced by the different nanomaterials and nanoparticles tested. Only zebrafish larvae exposed to free Au nanoparticles showed a different response to anti-MT antibody. In fact, the immunolocalization analysis highlighted an increase of the metallothioneins synthesis.
Alzheimer's disease is one of the most common causes of death worldwide, with poor treatment options. A tissue landmark of Alzheimer's disease is accumulation of the anomalous protein amyloid-β in ...specific brain areas. Whether inflammation is an effect of amyloid-β on the Alzheimer's disease brain, or rather it represents a cause for formation of amyloid plaques and intracellular tangles remains a subject of debate. TNFSF10, a proapoptotic cytokine of the TNF superfamily, is a mediator of amyloid-β neurotoxicity. Here, we demonstrate that blocking TNFSF10 by administration of a neutralizing monoclonal antibody could attenuate the amyloid-β-induced neurotoxicity in a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). The effects of TNFSF10 neutralization on either cognitive parameters, as well as on the expression of TNFSF10, amyloid-β, inflammatory mediators and GFAP were studied in the hippocampus of 3xTg-AD mice. Treatment with the TNFSF10 neutralizing antibody resulted in dramatic improvement of cognitive parameters, as assessed by the Morris water maze test and the novel object recognition test. These results were correlated with decreased protein expression of TNFSF10, amyloid-β, inflammatory mediators and GFAP in the hippocampus. Finally, neutralization of TNFSF10 results in functional improvement and restrained immune/inflammatory response in the brain of 3xTg-AD mice in vivo. Thus, it is plausible to regard the TNFSF10 system as a potential target for efficacious treatment of amyloid-related disorders.
Common sole (
Solea solea
) is often the subject of fraudulent species substitutions in processed products because of their excellent organoleptic characteristics and high commercial interest. ...COIBar-RFLP, a molecular strategy-coupling Cytochrome Oxidase I (COI) DNA barcoding with the consolidated methodology of restriction fragment length polymorphism analysis (RFLP), was applied to search for restriction enzyme polymorphisms useful to discriminate among potential flatfish substitutes of common sole. Seven flatfish species belonging to Soleidae, Bothidae, and Citharidae families were used to construct a reference barcode library of COI sequences. The flatfish species were simultaneously discriminated through specific digestion profiles obtained with the restriction enzyme
Msp
I. We tested the efficacy of COIBar-RFLP on 13 frozen fillets labeled as common sole purchased in local fish markets. These fillets were found to contain three species,
S. solea, Solea senegalensis
, and
Arnoglossus laterna
, successfully discriminated by COIBar-RFLP, demonstrating that this method is a rapid and simple sequencing-free molecular approach for these fish species identification in processed seafood products.
During diabetic retinopathy (DR) progression, the retina undergoes various metabolic changes, including hypoxia-signalling cascade induction in the cells of retinal pigmented epithelium (RPE). The ...overexpression of hypoxic inducible factors causes transcription of many target genes including vascular endothelial growth factor (VEGF). The RPE cells form the outer blood retinal barrier (oBRB), a specialized structure that regulates ions and metabolites flux into the retina to maintain a suitable quality of its extracellular microenvironment. VEGF worsens retinal condition since its secretion from the basolateral compartment of RPE cells compromises the barrier’s integrity and induces choroidal neovascularization. In this work, we hypothesized that PACAP prevents the damage to oBRB and controls choroidal neovascularization through the induction of ADNP. Firstly, we demonstrated that ADNP is expressed in Streptozotocin (STZ)-induced diabetic animals. To validate our hypothesis, we cultured endothelial cells (H5V) forming vessels-like structures, in a conditioned medium (CM) derived from ARPE-19 cells exposed to hyperglycaemic/hypoxic insult, containing a known VEGF concentration. The involvement of PACAP-ADNP axis on oBRB integrity was evaluated through the measurement of trans-epithelial-electrical resistance and permeability assay performed on ARPE cell monolayer cultured in CM and by analysing the expression of two tight junction forming proteins, ZO1 and occludin. By culturing H5V in CM, we demonstrated that PACAP-ADNP axis counteracted vessels-like structures formation promoted by VEGF. In conclusion, the results suggested a primary role of PACAP/ADNP axis in preventing oBRB damage and in controlling aberrant choroidal neovascularization induced by VEGF secreted from RPE cells exposed to hyperglycaemia/hypoxic insult in DR.
•PACAP/ADNP axis prevents blood retinal barrier damage made by VEGF secreted from pigmented epithelium under hyperglycemia.•In diabetic retinopathy, PACAP/ADNP axis controls neovascularization induced by VEGF secreted from pigmented epithelium.•PACAP/ADNP axis keep tight junctions between retinal pigmented epithelial cells exposed to hyperglycemic/ hypoxic insult.
•Halloysite clay nanotubes HNT-N,-U,-P,-S were analyzed by in vitro cell tests.•MTT and CBMN assays were used to test HNTs cellular and genomic toxicity.•HNTs, with the in vitro test on mammalian ...cells, showed a good biocompatibility.•Only at the highest concentrations HNTs showed a moderate level of toxicity.•Caution is suggested for future use of large amounts of HNTs on human applications.
The biocompatibility of four different halloysite clay nanotubes (HNT-N,-U,-P,-S), from various deposits in the world, was evaluated. More precisely, we tested their cytogenetic effects through MTT assay (for cytotoxic effects), and CBMN assay (for genotoxic effects) on mammalian cell cultures (CHO, HeLa, and HepG2 cell lines). Our results indicated a generally high level of biocompatibility of these new-generation products, however at high concentrations, and long times of exposition, we observed a moderate level of toxicity. Thus, we suggest caution in the future use of great amounts of HNTs due to their effects on living organisms.
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