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.

Beneficial Effect of Heat-induced Antigen Retrieval in Immunocytochemical Detection of Intracellular Antigens in Alcohol-fixed Cell Samples.

Immunohistochemistry and immunocytochemistry (ICC) play an irreplaceable role in research and diagnostics. It is well known that antigen retrieval (AR) can, as a technique, have beneficial outcomes on immunohistochemistry results when using formalin-fixed, paraffin-embedded tissue samples. The main purpose of AR is to break protein crosslinks which are formed during formalin fixation. Although AR was originally designed for formalin-fixed, paraffin-embedded samples, the usefulness of AR in ICC has been described in previous studies. Cytologic samples are often fixed in alcohol-based fixatives which does not lead to the formation of crosslinks. Therefore, alcohol-fixed samples can be successfully immunostained without AR. We investigated the effect of heat-induced antigen retrieval (HIAR) on alcohol-fixed HEK293 cell line samples and patient cytologic samples from thyroid gland obtained by fine needle aspiration technique. We compared indirect 2-step ICC staining results performed according to the protocol with or without HIAR in citrate buffer pH 6 for several antibodies. Utilizing HIAR against intracellular antigens has beneficial effects. Therefore, more diluted antibodies can be used for satisfactory results. However, surface antigens were probably damaged by HIAR treatment. We demonstrated evident changes in cell surface topography after HIAR treatment by atomic force microscopy. Staining specificity of patient samples improves and background staining is reduced, allowing higher dilutions of primary antibody. Improving staining specificity is necessary for accurate diagnostics. Although we have shown the beneficial effect of HIAR for immunostaining intracellular antigens, proper staining protocol should be tested on appropriate controls for individual antibodies.

Cell Membrane Nanostructure is Altered by Heat-Induced Antigen Retrieval: A Possible Consequence for Immunocytochemical Detection of Membranous Antigens.

Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.

HT-29 and Caco2 Cell Lines Are Suitable Models for Studying the Role of Arachidonic Acid-Metabolizing Enzymes in Intestinal Cell Differentiation.

INTRODUCTION: Cytochrome (CYP) epoxygenases (CYP2C and CYP2J) and soluble epoxide hydrolase (sEH) participate in the metabolism of arachidonic acid and may also have a potential role in enterocyte differentiation. The first critical step in the study of intestinal cell differentiation is the determination of a suitable in vitro model, which must be as similar as possible to the conditions of a living organism. It is known that HT-29 and Caco2 cell lines derived from human colorectal carcinomas can differentiate into enterocyte-like cells in appropriate culture conditions. MATERIAL AND METHODS: We tested 4 different approaches of enterocyte-like differentiation and determined the most appropriate culture conditions for each model. Subsequently, the changes in the expression of CYP epoxygenases and sEH in undifferentiated and differentiated cells were measured by In-Cell ELISA. These results were compared with immunohistochemical profiles of expression of CYP epoxygenases and sEH in samples of human embryonic and fetal intestines as well as adult duodenum and colon. RESULTS: Our results show that sodium butyrate (NaBt)-differentiated HT-29 cells and spontaneously differentiated Caco2 cells resemble CYP epoxygenases and sEH profiles, corresponding with different types of intestines. CONCLUSION: Our study revealed that the most suitable models for the study of the role of CYP epoxygenases and sEH expression in differentiation of intestinal epithelium are NaBt-differentiated HT-29 cells and spontaneously differentiated Caco2 cells.

Raman imaging of cellular uptake and studies of silver nanoparticles effect in BJ human fibroblasts cell lines.

Silver nanoparticles (AgNPs) have been widely studied for their beneficial antimicrobial effect and have been considered by some to be a safe ingredient, as penetration of metal nanoparticles through the skin in vivo has not been proven. However, AgNPs are becoming a commonly applied nanomaterial for surface modifications of medical products which come into contact with damaged skin. In our experiments, we tested two commercially available AgNPs samples manufactured by electrolysis. AFM was used to characterize tested AgNPs morphology and their mean particle size which was assessed as 30.6nm and 20.4nm. An important mechanism of AgNPs cytotoxicity is generation of reactive oxygen species (ROS), chemically reactive species containing oxygen. Although ROS occur in cell metabolism naturally, their overproduction can induce oxidative stress - imbalance between production and antioxidant defenses. This can be associated with cytotoxicity and DNA damage. Conventional in vitro tests were used to evaluate the cytotoxic potential and DNA damage in BJ human fibroblasts cell lines. We found that both tested AgNPs samples induced ROS generation and caused the DNA damage in fibroblasts. One of the key concerns about the association with cytotoxic or genotoxic responses of nanoparticles is the capability of these materials to penetrate through cellular membrane. Cellular uptake studies were performed using Raman imaging as a label-free microscopic technique. In combination with a univariate image analysis, results demonstrate cellular uptake and distribution of the AgNPs which were taken up by BJ cells within 24h of incubation in a growth medium. The study demonstrates the potential of Raman imaging to unambiguously identify and localize AgNPs in fixed cells.

Nuclear myosin I regulates cell membrane tension.

Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension.

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.

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.

The application of antimicrobial photodynamic therapy on S. aureus and E. coli using porphyrin photosensitizers bound to cyclodextrin.

Photodynamic therapy is usually used against malignant and non-malignant tumors. Nowadays, due to resistance of bacterial strains, we are looking for a new antimicrobial strategy to destroy bacteria with minimal invasive consequences. The worldwide increase in antibiotic resistance among different classes of gram-positive and gram-negative bacteria has led to the search for alternative anti-microbial therapies such as antimicrobial PDT (aPDT). Development antimicrobial technology combines a nontoxic compound, called photosensitizer, visible light of the appropriate wavelength, and the generation of reactive oxygen species. In this work, the photosensitizers TMPyP and ZnTPPS4 are investigated for photodynamic and antimicrobial photodynamic therapy. We tested these two porphyrins on two cell lines and two bacterial strains to compare effectiveness. In addition, we applied photosensitizers bound in the complex created with hp-ß-cyclodextrin. The light-emitting diodes were used at the doses 0, 1, 5, 10 J/cm(2) for cells and 0, 150 J/cm(2) for bacteria. Tested concentrations for cells and microbes were from 0.5 to 50 µM and from 0.78 to 100 µM, respectively. From this work it can be concluded that TMPyP is a promising compound both in aPDT and in PDT, particularly in contrast to ZnTPPS4, which was efficient only in PDT. Furthermore, the eradication of gram-positive bacteria is possible only with higher concentrations of ZnTPPS4.

The effect of photodynamic treatment on the morphological and mechanical properties of the HeLa cell line.

High resolution imaging of biological structures and changes induced by various agents such as drugs and toxins is commonly performed by fluorescence and electron microscopy (EM). Although high-resolution imaging is possible with EM, the requirements for fixation and staining of samples for image contrast severely limits the study of living organisms. Atomic force microscopy (AFM), on the other hand, is capable of simultaneous nanometer spatial resolution and piconewton force detection, allowing detailed study of cell surface morphology and monitoring cytomechanical information. We present a method that images and studies mechanically characterized cells using AFM. We used a HeLa cell line (cervix carcinoma cell), which is sensitive to photodynamic treatment (PDT); growth media as a scanning surrounding; atomic force microscopy NT-MDT Aura for cytomechanical measurement; and scanning electron microscope Hitachi Su 6600 for control images of the cells. The modulus of elasticity for intact and photodynamically damaged cells can indicate mechanical changes to the main properties of cells. Cell elasticity changes can provide information on the degree or value of cell damage, for example after PDT. Measurements were carried out on approximately sixty cells, including three independent experiments on a control group and on sixty cells in a photodamaged group. Cells before PDT show higher elasticity: the median of Young´s modulus on the nucleus was 35.283 kPa and outside of the nucleus 107.442 kPa. After PDT, the median of Young's modulus on the nucleus was 61.144 kPa and outside of the nucleus was 193.605 kPa.