Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-ß-cyclodextrin (methyl-ß-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

Effects of physicochemical properties of TiO2 nanomaterials for pulmonary inflammation, acute phase response and alveolar proteinosis in intratracheally exposed mice.

Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO2 in hazard assessment might be challenging because of variation in physicochemical properties. We exposed C57BL/6 J mice to a single dose of four anatase TiO2 NMs with various sizes and shapes by intratracheal instillation and assessed the pulmonary toxicity 1, 3, 28, 90 or 180 days post-exposure. The quartz DQ12 was included as benchmark particle. Pulmonary responses were evaluated by histopathology, electron microscopy, bronchoalveolar lavage (BAL) fluid cell composition and acute phase response. Genotoxicity was evaluated by DNA strand break levels in BAL cells, lung and liver in the comet assay. Multiple regression analyses were applied to identify specific TiO2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO2 NMs induced similar inflammatory responses when surface area was used as dose metrics, although inflammatory and acute phase response was greatest and more persistent for the TiO2 tube. Similar histopathological changes were observed for the TiO2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO2 NMs indicated that rutile TiO2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO2 NMs are important predictors for the observed pulmonary effects of TiO2 NMs.

Nanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer.

Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane's disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.

Factor Xa dimerization competes with prothrombinase complex formation on platelet-like membrane surfaces.

Exposure of phosphatidylserine (PS) molecules on activated platelet membrane surface is a crucial event in blood coagulation. Binding of PS to specific sites on factor Xa (fXa) and factor Va (fVa) promotes their assembly into a complex that enhances proteolysis of prothrombin by approximately 105. Recent studies demonstrate that both soluble PS and PS-containing model membranes promote formation of inactive fXa dimers at 5 mM Ca²âº. In the present study, we show how competition between fXa dimerization and prothrombinase formation depends on Ca²âº and lipid membrane concentrations. We used homo-FRET measurements between fluorescein-E-G-R-chloromethylketone (CK)-Xa [fXa irreversibly inactivated by alkylation of the active site histidine residue with FEGR (FEGR-fXa)] and prothrombinase activity measurements to reveal the balance between fXa dimer formation and fXa-fVa complex formation. Changes in FEGR-fXa dimer homo-FRET with addition of fVa to model-membrane-bound FEGR-fXa unambiguously demonstrated that formation of the FEGR-fXa-fVa complex dissociated the dimer. Quantitative global analysis according to a model for protein interaction equilibria on a surface provided an estimate of a surface constant for fXa dimer dissociation (K(fXa×fXa)(d, σ)) approximately 10-fold lower than K(fXa×fVa)(d,σ) for fXa-fVa complex. Experiments performed using activated platelet-derived microparticles (MPs) showed that competition between fXa dimerization and fXa-fVa complex formation was even more prominent on MPs. In summary, at Ca²âº concentrations found in the maturing platelet plug (2-5 mM), fVa can compete fXa off of inactive fXa dimers to significantly amplify thrombin production, both because it releases dimer inhibition and because of its well-known cofactor activity. This suggests a hitherto unanticipated mechanism by which PS-exposing platelet membranes can regulate amplification and propagation of blood coagulation.

Ca2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation.

Calcium (Ca2+) plays a pivotal role in cellular and organismal physiology. The Ca2+ ion has an intermediate protein-binding affinity and thus it can serve as an on/off switch in the regulation of different biochemical processes. The serum level of ionized Ca2+ is regulated with normal ionized Ca2+ being in the range 1.10-1.3 mM. Hypocalcaemia (free Ca2+<1.1 mM) in critically ill patients is commonly accompanied by haemostatic abnormalities, ranging from isolated thrombocytopenia to complex defects such as disseminated intravascular coagulation, commonly thought to be due to insufficient functioning of anticoagulation pathways. A small amount of fXa (Factor Xa) produced by Factor VIIa and exposed tissue factor is key to initiating blood coagulation by producing enough thrombin to induce the later stages of coagulation. fXa must bind to PS (phosphatidylserine)-containing membranes to produce thrombin at a physiologically significant rate. In the present study, we show that overall fXa activity on PS-containing membranes is sharply regulated by a 'Ca2+ switch' centred at 1.16 mM, below which fXa is active and above which fXa forms inactive dimers on PS-exposing membranes. Our data lead to a mathematical model that predicts the variation of fXa activity as a function of both Ca2+ and membrane concentrations. Because the critical Ca2+ concentration is at the lower end of the normal plasma ionized Ca2+ concentration range, we propose a new regulatory mechanism by which local Ca2+ concentration switches fXa from an intrinsically active form to a form requiring its cofactor [fVa (Factor Va)] to achieve significant activity.

Stable oligomeric clusters of gold nanoparticles: preparation, size distribution, derivatization, and physical and biological properties.

Reducing dilute aqueous HAuCl4 with NaSCN under alkaline conditions produces 2-3 nm diameter yellow nanoparticles without the addition of extraneous capping agents. We here describe two very simple methods for producing highly stable oligomeric grape-like clusters (oligoclusters) of these small nanoparticles. The oligoclusters have well-controlled diameters ranging from ∼5 to ∼30 nm, depending mainly on the number of subunits in the cluster. Our first ["delay-time"] method controls the size of the oligoclusters by varying from seconds to hours the delay time between making the HAuCl4 alkaline and adding the reducing agent, NaSCN. Our second ["add-on"] method controls size by using yellow nanoparticles as seeds onto which varying amounts of gold derived from "hydroxylated gold", Na(+)[Au(OH4-x)Clx](-), are added-on catalytically in the presence of NaSCN. Possible reaction mechanisms and a simple kinetic model fitting the data are discussed. The crude oligocluster preparations have narrow size distributions, and for most purposes do not require fractionation. The oligoclusters do not aggregate after ∼300-fold centrifugal-filter concentration, and at this high concentration are easily derivatized with a variety of thiol-containing reagents. This allows rare or expensive derivatizing reagents to be used economically. Unlike conventional glutathione-capped nanoparticles of comparable gold content, large oligoclusters derivatized with glutathione do not aggregate at high concentrations in phosphate-buffered saline (PBS) or in the circulation when injected into mice. Mice receiving them intravenously show no visible signs of distress. Their sizes can be made small enough to allow their excretion in the urine or large enough to prevent them from crossing capillary basement membranes. They are directly visible in electron micrographs without enhancement, and can model the biological fate of protein-like macromolecules with controlled sizes and charges. The ease of derivatizing the oligoclusters makes them potentially useful for presenting pharmacological agents to different tissues while controlling escape of the reagents from the circulation.

An elevated rate of whole-genome duplications in cancers from Black patients.

In the United States, Black individuals have higher rates of cancer mortality than any other racial group. Here, we examine chromosome copy number changes in cancers from more than 1800 self-reported Black patients. We find that tumors from self-reported Black patients are significantly more likely to exhibit whole-genome duplications (WGDs), a genomic event that enhances metastasis and aggressive disease, compared to tumors from self-reported white patients. This increase in WGD frequency is observed across multiple cancer types, including breast, endometrial, and lung cancer, and is associated with shorter patient survival. We further demonstrate that combustion byproducts are capable of inducing WGDs in cell culture, and cancers from self-reported Black patients exhibit mutational signatures consistent with exposure to these carcinogens. In total, these findings identify a type of genomic alteration that is associated with environmental exposures and that may influence racial disparities in cancer outcomes.

Particulate matter constituents trigger the formation of extracellular amyloid ß and Tau -containing plaques and neurite shortening in vitro.

Air pollution is an environmental factor associated with an increased risk of neurodegenerative diseases, such as Alzheimer's and Parkinson's, characterized by decreased cognitive abilities and memory. The limited models of sporadic Alzheimer's disease fail to replicate all pathological hallmarks of the disease, making it challenging to uncover potential environmental causes. Environmentally driven models of Alzheimer's disease are thus timely and necessary. We used live-cell confocal fluorescent imaging combined with high-resolution stimulated emission depletion (STED) microscopy to follow the response of retinoic acid-differentiated human neuroblastoma SH-SY5Y cells to nanomaterial exposure. Here, we report that exposure of the cells to some particulate matter constituents reproduces a neurodegenerative phenotype, including extracellular amyloid beta-containing plaques and decreased neurite length. Consistent with the existing in vivo research, we observed detrimental effects, specifically a substantial reduction in neurite length and formation of amyloid beta plaques, after exposure to iron oxide and diesel exhaust particles. Conversely, after exposure to engineered cerium oxide nanoparticles, the lengths of neurites were maintained, and almost no extracellular amyloid beta plaques were formed. Although the exact mechanism behind this effect remains to be explained, the retinoic acid differentiated SH-SY5Y cell in vitro model could serve as an alternative, environmentally driven model of neurodegenerative diseases, including Alzheimer's disease.

Phosphatidylserine-induced factor Xa dimerization and binding to factor Va are competing processes in solution.

A soluble, short chain phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to promote FXa dimerization (K(d) ~ 14 nM), and to enhance both the catalytic activity of FXa and the cofactor activity of FVa. In the presence of calcium, C6PS binds to two sites on FXa, one in the epidermal growth factor-like (EGF) domain and one in the catalytic domain; the latter interaction is sensitive to Na(+) binding and probably represents a protein recognition site. Here we ask whether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes. We monitored FXa activity at 5, 20, and 50 nM FXa while titrating with FVa in the presence of 400 µM C6PS and 3 or 5 mM Ca(2+) to show that the apparent K(d) of FVa-FXa interaction increased with an increase in FXa concentration at 5 mM Ca(2+), but the K(d) was only slightly affected at 3 mM Ca(2+). A mixture of 50 nM FXa and 50 nM FVa in the presence of 400 µM C6PS yielded both Xa homodimers and Xa·Va heterodimers, but no FXa dimers bound to FVa. A mutant FXa (R165A) that has reduced prothrombinase activity showed both weakened dimerization (K(d) ~ 147 nM) and weakened FVa binding (apparent K(d) values of 58, 92, and 128 nM for 5, 20, and 50 nM R165A FXa, respectively). Native gel electrophoresis showed that the GLA-EGF(NC) fragment of FXa (lacking the catalytic domain) neither dimerized nor formed a complex with FVa in the presence of 400 µM C6PS and 5 mM Ca(2+). Our results demonstrate that the dimerization site and FVa-binding site are both located in the catalytic domain of FXa and that these sites are linked thermodynamically.

Influence of cancerostatic perifosine on membrane fluidity of liposomes and different cell lines as measured by electron paramagnetic resonance.

AIM: To test whether membrane fluidity and its changes are important for the sensitivity of cells to the action of perifosine (OPP), a new anticancer drug targeting cell membrane and not DNA. METHOD: Influence of OPP on the membrane structure of OPP-resistant MCF7, and OPP-sensitive MT3 breast cancer cell lines, as well as of mouse fibroblasts (L929) cell lines, and model cells (liposomes) was investigated by electron paramagnetic resonance, using spin labeled derivative of OPP (P5) and 5-doxylpalmitoyl methylester (MeFASL(10,3)) as spin probes. RESULTS: OPP increased membrane fluidity of all cell lines at concentrations higher than 50 µM (on the level of P≤0.05, t test). In cells, the differences were observed only by P5 and not by MeFASL(10,3). Average order parameter Seff decreased for about 12% in MCF7 and L929 and only for 8% in OPP-sensitive MT3 cells, showing that there was no correlation between membrane fluidity changes and sensitivity of cells to OPP. The only correlation we found was between OPP sensitivity and the cell growth rate. In liposomes, both spin probes were sensitive to the action of OPP. Seff decreased with increasing concentration of OPP. For MeFASL(10,3) a significant decrease was observed at 4 mol% OPP, while for P5 it was observed at 8 mol%. CONCLUSION: Influence of OPP on plasma membrane fluidity of breast cancer cells is not the determining factor in the sensitivity of cells to OPP.

Aerosol-Cell Exposure System Applied to Semi-Adherent Cells for Aerosolization of Lung Surfactant and Nanoparticles Followed by High Quality RNA Extraction.

Nanoparticle toxicity assessments have moved closer to physiological conditions while trying to avoid the use of animal models. An example of new in vitro exposure techniques developed is the exposure of cultured cells at the air-liquid interface (ALI), particularly in the case of respiratory airways. While the commercially available VITROCELL® Cloud System has been applied for the delivery of aerosolized substances to adherent cells under ALI conditions, it has not yet been tested on lung surfactant and semi-adherent cells such as alveolar macrophages, which are playing a pivotal role in the nanoparticle-induced immune response. OBJECTIVES: In this work, we developed a comprehensive methodology for coating semi-adherent lung cells cultured at the ALI with aerosolized surfactant and subsequent dose-controlled exposure to nanoparticles (NPs). This protocol is optimized for subsequent transcriptomic studies. METHODS: Semi-adherent rat alveolar macrophages NR8383 were grown at the ALI and coated with lung surfactant through nebulization using the VITROCELL® Cloud 6 System before being exposed to TiO2 NM105 NPs. After NP exposures, RNA was extracted and its quantity and quality were measured. RESULTS: The VITROCELL® Cloud system allowed for uniform and ultrathin coating of cells with aerosolized surfactant mimicking physiological conditions in the lung. While nebulization of 57 µL of 30 mg/mL TiO2 and 114 µL of 15 mg/mL TiO2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 µL.

Hemagglutinin fusion peptide mutants in model membranes: structural properties, membrane physical properties, and PEG-mediated fusion.

While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events.

Determination of the amount of micelles in alkylphospholipid liposome formulations with electron paramagnetic resonance method.

Alkylphospholipids (APLs) are physiologically active derivatives of lipids with high efficiency in the therapy of experimental human breast cancer. Therefore, they are usually used as liposome formulations, since the application of free, micellar APLs results in serious side effects, such as hemolysis. Since only rough upper estimates of micelle proportions were obtained until now, we developed a new method, based on the spectral decomposition of electron paramagnetic resonance (EPR) spectra. The EPR spectra of different spin-labeled mixed liposome-micelle samples were fitted with a fast, automatic fitting procedure. In this way, we achieved more accurate estimates of the relative proportion of micelles, in comparison to the previously used methods. We confirmed findings of previous studies, which showed that the amount of micelles in OPP liposome formulations increases with a decreasing amount of cholesterol. We analyzed liposome formulations with 29, 39, and 46 mol% of cholesterol and determined the relative proportion of micelles to be 20 ± 9, 11 ± 4, and 5 ± 2, respectively. This study confirms that the efficiency of OPP liposome formulations in experimental breast cancer therapy is not solely a consequence of the amount of micelles present in a liposome formulation, but stems from a propensity of liposomes to interact with cells.

Phosphatidylserine and phosphatidylethanolamine regulate the structure and function of FVIIa and its interaction with soluble tissue factor.

Cell membranes have important functions in many steps of the blood coagulation cascade, including the activation of factor X (FX) by the factor VIIa (FVIIa)-tissue factor (TF) complex (extrinsic Xase). FVIIa shares structural similarity with factor IXa (FIXa) and FXa. FIXa and FXa are regulated by binding to phosphatidylserine (PS)-containing membranes via their γ-carboxyglutamic acid-rich domain (Gla) and epidermal growth-factor (EGF) domains. Although FVIIa also has a Gla-rich region, its affinity for PS-containing membranes is much lower compared with that of FIXa and FXa. Research suggests that a more common endothelial cell lipid, phosphatidylethanolamine (PE), might augment the contribution of PS in FVIIa membrane-binding and proteolytic activity. We used soluble forms of PS and PE (1,2-dicaproyl-sn-glycero-3-phospho-l-serine (C6PS), 1,2-dicaproyl-sn-glycero-3-phospho-ethanolamine (C6PE)) to test the hypothesis that the two lipids bind to FVIIa jointly to promote FVIIa membrane binding and proteolytic activity. By equilibrium dialysis and tryptophan fluorescence, we found two sites on FVIIa that bound equally to C6PE and C6PS with Kd of ∼ 150-160 µM, however, deletion of Gla domain reduced the binding affinity. Binding of lipids occurred with greater affinity (Kd∼70-80 µM) when monitored by FVIIa proteolytic activity. Global fitting of all datasets indicated independent binding of two molecules of each lipid. The proteolytic activity of FVIIa increased by ∼50-100-fold in the presence of soluble TF (sTF) plus C6PS/C6PE. However, the proteolytic activity of Gla-deleted FVIIa in the presence of sTF was reduced drastically, suggesting the importance of Gla domain to maintain full proteolytic activity.

How to control fluorescent labeling of metal oxide nanoparticles for artefact-free live cell microscopy.

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles-the largest subpopulation of nanoparticles by industrial production and applications-and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials.

Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells.

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin-probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin-treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.

Characterization of blood coagulation dynamics and oxygenation in ex-vivo retinal vessels by fluorescence hyperspectral imaging.

Blood coagulation mechanisms forming a blood clot and preventing hemorrhage have been extensively studied in the last decades. Knowing the mechanisms behind becomes very important particularly in the case of blood vessel diseases. Real-time and accurate diagnostics accompanied by the therapy are particularly needed, for example, in diseases related to retinal vasculature. In our study, we employ for the first time fluorescence hyperspectral imaging (fHSI) combined with the spectral analysis algorithm concept to assess physical as well as functional information of blood coagulation in real-time. By laser-induced local disruption of retinal vessels to mimic blood leaking and subsequent coagulation and a proper fitting algorithm, we were able to reveal and quantify the extent of local blood coagulation through direct identification of the change of oxyhemoglobin concentration within few minutes. We confirmed and illuminated the spatio-temporal evolution of the essential role of erythrocytes in the coagulation cascade as the suppliers of oxygenated hemoglobin. By additional optical tweezers force manipulation, we showed immediate aggregation of erythrocytes at the coagulation site. The presented fluorescence-based imaging concept could become a valuable tool in various blood coagulation diagnostics as well as theranostic systems if coupled with the laser therapy.

Photocatalytic biocidal effect of copper doped TiO2 nanotube coated surfaces under laminar flow, illuminated with UVA light on Legionella pneumophila.

Legionella pneumophila can cause a potentially fatal form of humane pneumonia (Legionnaires' disease), which is most problematic in immunocompromised and in elderly people. Legionella species is present at low concentrations in soil, natural and artificial aquatic systems and is therefore constantly entering man-made water systems. The environment temperature for it's ideal growth range is between 32 and 42°C, thus hot water pipes represent ideal environment for spread of Legionella. The bacteria are dormant below 20°C and do not survive above 60°C. The primary method used to control the risk from Legionella is therefore water temperature control. There are several other effective treatments to prevent growth of Legionella in water systems, however current disinfection methods can be applied only intermittently thus allowing Legionella to grow in between treatments. Here we present an alternative disinfection method based on antibacterial coatings with Cu-TiO2 nanotubes deposited on preformed surfaces. In the experiment the microbiocidal efficiency of submicron coatings on polystyrene to the bacterium of the genus Legionella pneumophila with a potential use in a water supply system was tested. The treatment thus constantly prevents growth of Legionella pneumophila in presence of water at room temperature. Here we show that 24-hour illumination with low power UVA light source (15 W/m2 UVA illumination) of copper doped TiO2 nanotube coated surfaces is effective in preventing growth of Legionella pneumophila. Microbiocidal effects of Cu-TiO2 nanotube coatings were dependent on the flow of the medium and the intensity of UV-A light. It was determined that tested submicron coatings have microbiocidal effects specially in a non-flow or low-flow conditions, as in higher flow rates, probably to a greater possibility of Legionella pneumophila sedimentation on the coated polystyrene surfaces, meanwhile no significant differences among bacteria reduction was noted regarding to non or low flow of medium.