Hyperoside as a UV Photoprotective or Photostimulating Compound-Evaluation of the Effect of UV Radiation with Selected UV-Absorbing Organic Compounds on Skin Cells.

Ultraviolet (UV) radiation is a non-ionizing radiation, which has a cytotoxic potential, and it is therefore necessary to protect against it. Human skin is exposed to the longer-wavelength components of UV radiation (UVA and UVB) from the sun. In the present paper, we focused on the study of eight organic UV-absorbing compounds: astragalin, beta-carotene, 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, hyperoside, 3-(4-methylbenzylidene)camphor, pachypodol, and trans-urocanic acid, as possible protectives of skin cells against UVA and UVB radiation. Their protective effects on skin cell viability, ROS production, mitochondrial membrane potential, liposomal permeability, and DNA integrity were investigated. Only some of the compounds studied, such as trans-urocanic acid and hyperoside, had a significant effect on the examined hallmarks of UV-induced cell damage. This was also confirmed by an atomic force microscopy study of morphological changes in HaCaT cells or a study conducted on a 3D skin model. In conclusion, hyperoside was found to be a very effective UV-protective compound, especially against UVA radiation. Commonly used sunscreen compounds such as 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, and 3-(4-methylbenzylidene)camphor turned out to be only physical UV filters, and pachypodol with a relatively high absorption in the UVA region was shown to be more phototoxic than photoprotective.

Time- and dose-dependent seeding tendency of exogenous tau R2 and R3 aggregates in cells.

Tauopathies are a group of neurodegenerative diseases categorised into three types, 3R, 4R, or 3R+4R (mixed) tauopathies, based on the tau isoforms that make up the aberrant filaments. It is supposed that all six tau isoforms share functional characteristics. However, differences in the neuropathological features associated with different tauopathies offer the possibility that disease progression and tau accumulation may vary depending on the isoform composition. The presence or absence of repeat 2 (R2) in the microtubule-binding domain defines the type of isoform, which might influence tau pathology associated with a particular tau isoform. Therefore, our study aimed to identify the differences in the seeding propensities of R2 and repeat 3 (R3) aggregates using HEK293T biosensor cells. We show that the seeding induced by R2 was generally higher than by R3 aggregates, and lower concentrations of R2 aggregates are sufficient to induce seeding. Next, we found that both R2 and R3 aggregates dose-dependently increased triton-insoluble Ser262 phosphorylation of native tau, which is only visible in cells seeded with higher concentrations (12.5 nM or 100 nM) of R2 and R3 aggregates, despite the seeding by the lower concentrations of R2 aggregates after 72 h. However, the accumulation of triton-insoluble pSer262 tau was visible earlier in cells induced with R2 than in R3 aggregates. Our findings suggest that the R2 region may contribute to the early and enhanced induction of tau aggregation and define the difference in disease progression and neuropathology of 4R tauopathies.

Photodynamic therapy: Innovative approaches for antibacterial and anticancer treatments.

Photodynamic therapy is an alternative treatment mainly for cancer but also for bacterial infections. This treatment dates back to 1900 when a German medical school graduate Oscar Raab found a photodynamic effect while doing research for his doctoral dissertation with Professor Hermann von Tappeiner. Unexpectedly, Raab revealed that the toxicity of acridine on paramecium depends on the intensity of light in his laboratory. Photodynamic therapy is therefore based on the administration of a photosensitizer with subsequent light irradiation within the absorption maxima of this substance followed by reactive oxygen species formation and finally cell death. Although this treatment is not a novelty, there is an endeavor for various modifications to the therapy. For example, selectivity and efficiency of the photosensitizer, as well as irradiation with various types of light sources are still being modified to improve final results of the photodynamic therapy. The main aim of this review is to summarize anticancer and antibacterial modifications, namely various compounds, approaches, and techniques, to enhance the effectiveness of photodynamic therapy.

The Potential for Genotoxicity, Mutagenicity and Endocrine Disruption in Triclosan and Triclocarban Assessed through a Combination of In Vitro Methods.

Triclosan and Triclocarban, preservatives widely used in cosmetics and other consumer products, underwent evaluation using a battery of new-approach methodologies in vitro (NAMs). Specifically, the Microplate Ames Test (MPF™ Test, Xenometrix, Allschwil, Switzerland) was employed to assess mutagenicity, the Comet assay in vitro on the HaCat cell line and the Mammalian Chromosome Aberration Test were utilized to evaluate genotoxicity, and the XenoScreen® YES/YAS assay was applied to investigate endocrine disruption. The chemicals did not exhibit any positive responses for mutagenicity. However, the mammalian chromosome aberration test identified both chemicals as being positive for genotoxicity at 10 µg/mL. In the Comet assay, the percentage of DNA in the tail significantly increased in a concentration-dependent manner (at 5 and 10 µg/mL for Triclosan, at 2.5, 5, and 10 µg/mL for Triclocarban). The positive response depended on the increasing concentration and the duration of exposure. Triclosan, but not Triclocarban in any of the endocrine assays performed, indicated a potential for endocrine activity in the anti-estrogenic and anti-androgenic assays. The positive in vitro results detected were obtained for concentrations relevant to final products. The alarming findings obtained with the use of new-approach methodologies (NAMs) justify the current precautionary regulatory approach, limiting the use of these preservatives.

Tau R2 and R3 are essential regions for tau aggregation, seeding and propagation.

Tauopathies are characterised by intracellular deposits of fibrillar tau tangles. However, the interneuronal spread of pathological tau species precedes the development of major tau burdens. Two amyloid motifs, VQIINK in repeat 2 and VQIVYK in repeat 3, of tau repeat domain, assemble into ß-sheet-rich fibrils on their own but alone do not form seed-competent fibrils. In contrast, the entire R3 region self-aggregates and forms seed-competent fibrils. Our study aimed to identify the minimal regions in the tau repeat domain that define seeding and its impact on intracellular tau phosphorylation and aggregation. Using peptides of individual repeats, we show that R2, like R3, forms seed-competent fibrils when assembled in the presence of heparin. However, R3, but not R2, forms seed-competent fibrils when assembled without heparin, even though both R2 and R3 have identical N-terminal hexapeptide and cysteine residue sequences. Moreover, cysteine to alanine substitution in R3 abrogates its self-aggregation and seeding potency. Tau RD P301S biosensor cells and Tau P301L (0N4R)-expressing HEK293 cells seeded with R2 and R3 fibrils show the induction of pathological phosphorylation of tau at Ser262/Ser396/Ser404 positions and oligomerisation of native tau. Protein fractions of biosensor cells seeded with R2 and R3 fibrils reseed endogenous tau aggregation when introduced into a fresh set of biosensor cells. Our findings suggest that R3 may be the minimal region for pathological seed generation under physiological conditions, whereas R2 might need polyanionic cofactors to generate pathogenic seeds. Lastly, R2 and R3 fibrils induce template-induced misfolding and pathological hyperphosphorylation of intracellular tau, making intracellular tau seed-competent.

Biological Evaluation of Photodynamic Effect Mediated by Nanoparticles with Embedded Porphyrin Photosensitizer.

Clinically approved photodynamic therapy (PDT) is a minimally invasive treatment procedure that uses three key components: photosensitization, a light source, and tissue oxygen. However, the photodynamic effect is limited by both the photophysical properties of photosensitizers as well as their low selectivity, leading to damage to adjacent normal tissue and/or inadequate biodistribution. Nanoparticles (NPs) represent a new option for PDT that can overcome most of the limitations of conventional photosensitizers and can also promote photosensitizer accumulation in target cells through enhanced permeation and retention effects. In this in vitro study, the photodynamic effect of TPP photosensitizers embedded in polystyrene nanoparticles was observed on the non-tumor NIH3T3 cell line and HeLa and G361 tumor cell lines. The efficacy was evaluated by viability assay, while reactive oxygen species production, changes in membrane mitochondrial potential, and morphological changes before and after treatment were imaged by atomic force microscopy. The tested nanoparticles with embedded TPP were found to become cytotoxic only after activation by blue light (414 nm) due to the production of reactive oxygen species. The photodynamic effect observed in this evaluation was significantly higher in both tumor lines than the effect observed in the non-tumor line, and the resulting phototoxicity depended on the concentration of photosensitizer and irradiation time.

Intracellular Trafficking of Cationic Carbon Dots in Cancer Cell Lines MCF-7 and HeLa-Time Lapse Microscopy, Concentration-Dependent Uptake, Viability, DNA Damage, and Cell Cycle Profile.

Fluorescent carbon dots (CDs) are potential tools for the labeling of cells with many advantages such as photostability, multicolor emission, small size, rapid uptake, biocompatibility, and easy preparation. Affinity towards organelles can be influenced by the surface properties of CDs which affect the interaction with the cell and cytoplasmic distribution. Organelle targeting by carbon dots is promising for anticancer treatment; thus, intracellular trafficking and cytotoxicity of cationic CDs was investigated. Based on our previous study, we used quaternized carbon dots (QCDs) for treatment and monitoring the behavior of two human cancer cell MCF-7 and HeLa lines. We found similarities between human cancer cells and mouse fibroblasts in the case of QCDs uptake. Time lapse microscopy of QCDs-labeled MCF-7 cells showed that cells are dying during the first two hours, faster at lower doses than at higher ones. QCDs at a concentration of 100 µg/mL entered into the nucleus before cellular death; however, at a dose of 200 µg/mL, blebbing of the cellular membrane occurred, with a subsequent penetration of QCDs into the nuclear area. In the case of HeLa cells, the dose-depended effect did not happen; however, the labeled cells were also dying in mitosis and genotoxicity occurred nearly at all doses. Moreover, contrasted intracellular compartments, probably mitochondria, were obvious after 24 h incubation with 100 µg/mL of QCDs. The levels of reactive oxygen species (ROS) slightly increased after 24 h, depending on the concentration, thus the genotoxicity was likely evoked by the nanomaterial. A decrease in viability did not reach IC 50 as the DNA damage was probably partly repaired in the prolonged G0/G1 phase of the cell cycle. Thus, the defects in the G2/M phase may have allowed a damaged cell to enter mitosis and undergo apoptosis. The anticancer effect in both cell lines was manifested mainly through genotoxicity.

Privacy-preserving solution for vehicle parking services complying with EU legislation.

Today, many modern cities adopt online smart parking services as best practices. Citizens can easily access these services using their smartphones or the infotainment panels in their cars. These services' primary objective is to give drivers the ability to quickly identify free parking slots, which should reduce parking time, save fuel, and relieve traffic in urban areas. However, the privacy offered by these services should be comparable to that of the standard paper-based parking solutions offered by parking ticket machines. On the other hand, a privacy-preserving smart parking service's design may raise a number of issues, including how to prevent double or multiple uses of parking tickets, how to prevent user tracking and profiling, how to revoke malicious users, how to handle data statistics without violating users' privacy, and how to comply with regulations like the General Data Protection Regulation (GDPR). In this article, we present multidisciplinary research on a comprehensive vehicle parking system that protects users' privacy. The research includes a range of topics, from the examination of regulatory compliance to the design of privacy-preserving parking registration and vehicle parking services to the implementation of privacy-preserving parking data processing features for data analysts. We provide a security analysis of our concept as well as several experimental results.

Antitumour drugs targeting tau R3 VQIVYK and Cys322 prevent seeding of endogenous tau aggregates by exogenous seeds.

Emerging experimental evidence suggests tau pathology spreads between neuroanatomically connected brain regions in a prion-like manner in Alzheimer's disease (AD). Tau seeding, the ability of prion-like tau to recruit and misfold naïve tau to generate new seeds, is detected early in human AD brains before the development of major tau pathology. Many antitumour drugs have been reported to confer protection against neurodegeneration, supporting the repurposing of approved and experimental or investigational oncology drugs for AD therapy. In this study, we evaluated whether antitumour drugs that abrogate the generation of seed-competent aggregates of tau Repeat 3 (R3) domain peptides can prevent tau seeding and toxicity in Tau-RD P301S FRET Biosensor cells and Caenorhabditis elegans. We demonstrate that drugs that interact with the N-terminal VQIVYK or the C-terminal region housing the Cys322 prevent R3 dimerisation, abolishing the generation of prion-like R3 seeds. Preformed R3 seeds (fibrils) capped with, or R3 seeds formed in the presence of VQIVYK- or Cys322-targeting drugs have a reduced potency to cause aggregation of naïve tau in biosensor cells and protect worms from aggregate toxicity. These findings indicate that VQIVYK- or Cys322-targeting drugs may act as prophylactic agents against tau seeding.

Synthesis of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines and their photodynamic properties in the human skin melanoma cell line G361.

A small series of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines was synthesized from easily accessible 1-phenyl-1H-pyrazol-3-ol via 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine and 7-iodo-4-methyl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine intermediates and their subsequent use in palladium catalyzed Buchwald-Hartwig cross-coupling reaction with various anilines. Majority of the compounds were not significantly cytotoxic to melanoma G361 cells in the dark up to 10 µM concentration, but their activity could be increased by irradiation with visible blue light (414 nm). The most active compound 10 possessed EC50 values of 3.5, 1.6 and 0.9 µM in cells irradiated with 1, 5 and 10 J/cm2, respectively. The treatment caused generation of reactive oxygen species in cells and extensive DNA damage, documented by the comet assay and by detection of phosphorylated histone H2A.X, followed by apoptotic cell death. Our results suggest that N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines could serve as a potential source of photosensitizing compounds with anticancer activities.

Self-Targeting of Carbon Dots into the Cell Nucleus: Diverse Mechanisms of Toxicity in NIH/3T3 and L929 Cells.

It is important to understand the nanomaterials intracellular trafficking and distribution and investigate their targeting into the nuclear area in the living cells. In our previous study, we firstly observed penetration of nonmodified positively charged carbon dots decorated with quaternary ammonium groups (QCDs) into the nucleus of mouse NIH/3T3 fibroblasts. Thus, in this work, we focused on deeper study of QCDs distribution inside two healthy mouse NIH/3T3 and L929 cell lines by fluorescence microspectroscopy and performed a comprehensive cytotoxic and DNA damage measurements. Real-time penetration of QCDs across the plasma cell membrane was recorded, concentration dependent uptake was determined and endocytic pathways were characterized. We found out that the QCDs concentration of 200 µg/mL is close to saturation and subsequently, NIH/3T3 had a different cell cycle profile, however, no significant changes in viability (not even in the case with QCDs in the nuclei) and DNA damage. In the case of L929, the presence of QCDs in the nucleus evoked a cellular death. Intranuclear environment of NIH/3T3 cells affected fluorescent properties of QCDs and evoked fluorescence blue shifts. Studying the intracellular interactions with CDs is essential for development of future applications such as DNA sensing, because CDs as DNA probes have not yet been developed.

Photodynamic effect of TPP encapsulated in polystyrene nanoparticles toward multi-resistant pathogenic bacterial strains: AFM evaluation.

Photodynamic inactivation (PDI) is a promising approach for the efficient killing of pathogenic microbes. In this study, the photodynamic effect of sulfonated polystyrene nanoparticles with encapsulated hydrophobic 5,10,15,20-tetraphenylporphyrin (TPP-NP) photosensitizers on Gram-positive (including multi-resistant) and Gram-negative bacterial strains was investigated. The cell viability was determined by the colony forming unit method. The results showed no dark cytotoxicity but high phototoxicity within the tested conditions. Gram-positive bacteria were more sensitive to TPP-NPs than Gram-negative bacteria. Atomic force microscopy was used to detect changes in the morphological properties of bacteria before and after the PDI treatment.

The effect of two porphyrine photosensitizers TMPyP and ZnTPPS4 for application in photodynamic therapy of cancer cells in vitro.

Photodynamic therapy (PDT) is one of the treatments for cancer. This therapy uses a combination of a photosensitizer (PS), light irradiation, and oxygen O2, which is converted to cytotoxic 1O2 and other forms of reactive oxygen species (ROS), causing selective damage to the target tissue. In this work, we studied effect of two porphyrin photosensitizers TMPyP and ZnTPPS4 at three different concentrations (0.25, 0.5, 5µM) after two irradiation doses (5 and 25 J/cm2). Photodynamic efect of TMPyP and ZnTPPS4 were confirmed by a battery of in vitro tests including MTT, reactive oxygen species (ROS) production and mitochondrial membrane potential test (MMP). Morphological changes of the cells before and after treatment were imaged by atomic force microscopy (AFM). The most effective combination of irradiation dose and concentration for both PSs showed a concentration of 5 µM and a irradiation dose of 25 J/cm2 in both cell cultures.

The effect of silver nanoparticles and silver ions on mammalian and plant cells in vitro.

Silver nanoparticles (AgNPs) are the most frequently applied nanomaterials. In our experiments, we tested AgNPs (size 27 nm) manufactured by the Tollens process. Physico-chemical methods (TEM, DLS, AFM and spectrophotometry) were used for characterization and imaging of AgNPs. The effects of AgNPs and Ag(+) were studied in two experimental models (plant and mammalian cells). Human keratinocytes (SVK14) and mouse fibroblasts (NIH3T3) cell lines were selected to evaluate the cytotoxicity and genotoxicity effect on mammalian cells. Higher sensitivity to AgNPs and Ag(+) was observed in NIH3T3 than in SVK14 cells. AgNPs accumulated in the nucleus of NIH3T3 cells, caused DNA damage and increased the number of apoptotic and necrotic cells. Three genotypes of Solanum spp. (S. lycopersicum cv. Amateur, S. chmielewskii, S. habrochaites) were selected to test the toxicity of AgNPs and Ag(+) on the plant cells. The highest values of peroxidase activity and lipid peroxidation were recorded after the treatment of S. habrochaites genotype with AgNPs. Increased ROS levels were likely the reason for observed damaged membranes in S. habrochaites. We found that the cytotoxic and genotoxic effects of AgNPs depend not only on the characteristics of nanoparticles, but also on the type of cells that are treated with AgNPs.

Effect of Porphyrin Sensitizer MgTPPS4 on Cytoskeletal System of HeLa Cell Line-Microscopic Study.

Metalloporphyrins are an important group of sensitizers with a porphyrin skeleton. Their photophysical properties are significantly affected by the nature of the central ion. In this work, we focus on the mechanical properties of a cervix carcinoma cell line which underwent photodynamic treatment (PDT) with MgTPPS4 photosensitzer. Atomic force microscopy alongside confocal microscopy was used to quantify and qualify the structural characteristics before and after PDT. Cells before PDT showed a fine actin network and higher elasticity with the median of Young modulus 12.2 kPa. After PDT, the median of Young modulus was 13.4 kPa and a large redistribution in the actin network was observed.

Reprint of: Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects. The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

Phthalocyanine-mediated Photodynamic Treatment of Tumoural and Non-tumoural cell lines.

This study deals with the use of cationic far-red absorbing photosensitizers (λ(max) ~740 nm) from the group of the phthalocyanines, in photodynamic therapy. The photosensitizers differed in their central atom, bearing either hydrogen, zinc or magnesium. These photosensitizers were tested in vitro on the tumour cell line HeLa (cervical cancer) and non-tumour cell line NIH3T3 (mouse fibroblast). The following tests were performed: measurement of reactive oxygen species production, viability testing, Comet assay and cell type detection (apoptotic, necrotic and living cells). The best results were achieved with zinc derivative at relatively low half-maximum inhibitory concentration (0.04 µM) and a total radiation dose of 15 J cm(-2).

Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects. The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

In vitro cytotoxicity analysis of doxorubicin-loaded/superparamagnetic iron oxide colloidal nanoassemblies on MCF7 and NIH3T3 cell lines.

One of the promising strategies for improvement of cancer treatment is based on magnetic drug delivery systems, thus avoiding side effects of standard chemotherapies. Superparamagnetic iron oxide (SPIO) nanoparticles have ideal properties to become a targeted magnetic drug delivery contrast probes, named theranostics. We worked with SPIO condensed colloidal nanocrystal clusters (MagAlg) prepared through a new soft biomineralization route in the presence of alginate as the polymeric shell and loaded with doxorubicin (DOX). The aim of this work was to study the in vitro cytotoxicity of these new MagAlg-DOX systems on mouse fibroblast and breast carcinoma cell lines. For proper analysis and understanding of cell behavior after administration of MagAlg-DOX compared with free DOX, a complex set of in vitro tests, including production of reactive oxygen species, comet assay, cell cycle determination, gene expression, and cellular uptake, were utilized. It was found that the cytotoxic effect of MagAlg-DOX system is delayed compared to free DOX in both cell lines. This was attributed to the different mechanism of internalization of DOX and MagAlg-DOX into the cells, together with the fact that the drug is strongly bound on the drug nanocarriers. We discovered that nanoparticles can attenuate or even inhibit the effect of DOX, particularly in the tumor MCF7 cell line. This is a first comprehensive study on the cytotoxic effect of DOX-loaded SPIO compared with free DOX on healthy and cancer cell lines, as well as on the induced changes in gene expression.