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Photocatalytic degradation of dichloroacetic acid (DCA) was studied in a continuous-flow set-up using a titanium microreactor with an immobilized double-layered TiO2 nanoparticle/nanotube film. Chloride ions, formed during the degradation process, negatively affect the photocatalytic efficiency and at a certain concentration (approximately 0.5 mM) completely stop the reaction in the microreactor. Two proposed mechanisms of inhibition with chloride ions, competitive adsorption and photogenerated-hole scavenging, have been proposed and investigated by adsorption isotherms and electron paramagnetic resonance (EPR) measurements. The results show that chloride ions block the DCA adsorption sites on the titania surface and reduce the amount of adsorbed DCA molecules. The scavenging effect of chloride ions during photocatalysis through the formation of chlorine radicals was not detected. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a pathogenic bacterium that can form biofilms in food processing plants, allowing the bacteria to survive despite the control measures applied. As the surface of the bacteria is covered with versatile polysaccharides and proteins, these influence the interactions of the bacterium with any surface. The unique properties and high stability of fungal proteins make them good candidates for the control of bacteria by targeting surface structures. We screened a group of fungal lectins and protease inhibitors from different fungal species, protein folds and known targets for their antibacterial and antibiofilm activity against model strains of Listeria innocua and Listeria monocytogenes. Several of them significantly decreased the viability of biofilm bacteria, but had no effect on bacterial growth parameters at 37°C and thus had no antibacterial activity. Fungal lectins significantly impaired biofilm development even at room temperature, which was attributed to exposure to lectins during adhesion. The tested fungal proteins also reduced biofilm development on biological model surfaces. The observed antibiofilm activity of fungal proteins suggests that they have the potential to modulate interactions between bacteria and/or between bacteria and surfaces, which could be used in the future to reduce surface contamination by Listeria., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

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.

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 TiO 2 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 TiO 2 and 114 μL of 15 mg/mL TiO 2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 μL.

Knowing the temperature distribution within the conducting walls of various multilayer-type materials is crucial for a better understanding of heat-transfer processes. This applies to many engineering fields, good examples being photovoltaics and microelectronics. In this work we present a novel fluorescence technique that makes possible the non-invasive imaging of local temperature distributions within a transparent, temperature-sensitive, co-doped Er:GPF1Yb0.5Er glass-ceramic with micrometer spatial resolution. The thermal imaging was performed with a high-resolution fluorescence microscopy system, measuring different focal planes along the z -axis. This ultimately enabled a precise axial reconstruction of the temperature distribution across a 500-µm-thick glass-ceramic sample. The experimental measurements showed good agreement with computer-modeled heat simulations and suggest that the technique could be adopted for the spatial analyses of local thermal processes within optically transparent materials. For instance, the technique could be used to measure the temperature distribution of intermediate, transparent layers of novel ultra-high-efficiency solar cells at the micron and sub-micron levels.

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.

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 TiO 2 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.

Tissue diseases and related disorders need to be first recognized using diagnostic methods and then later treated by therapeutic methods-a joint procedure called theranostics. One of the main challenges in the field of retinal therapies remains in the success of the treatment, typically improving the local metabolism, by sparing the surrounding tissue and with the immediate information of the laser effect. In our study, we present a concept for real-time controlled tissue theranostics on a proof-of-concept study capable of using a single tunable ps laser source (in terms of irradiance, fluence, and repetition rate), done on ex-vivo human retinal pigment epithelium. We have found autofluorescence intensity and lifetime imaging diagnostics very promising for the recognition and quantification of laser effects ranging from selective non-destructive molecular tissue modification to complete tissue ablation. The main novelty of our work presents the developed algorithm for optimized theranostics based on the model function used to quantify laser-induced tissue changes through the diagnostics descriptors, fluorescence lifetime and fluorescence intensity parameters. This approach, together with the operation of the single adaptable laser source, can serve as a new theranostics method in personalized medicine in the future not only limited to treat retinal diseases., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn-broken implant materials are still poorly understood on the subcellular and molecular level. Current immunohistochemical techniques lack spatial resolution and chemical sensitivity to investigate causal relations between material and biological response on submicron and even nanoscale. In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches. We have successfully characterized its chemical and biological impact on the periprosthetic tissue obtained at revision surgery of a fractured titanium-alloy modular neck of a patient with hip osteoarthritis. We applied a combination of photon, electron and ion beam micro-spectroscopy techniques, including hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris were found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosome. This may explain the indications of chronic inflammation from prior histologic examination. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. The functional and structural information obtained at molecular and subcellular level contributes to a better understanding of the macroscopic inflammatory processes observed in the tissue level. The established label-free correlative microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR.

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.

On a daily basis, people are exposed to a multitude of health-hazardous airborne particulate matter with notable deposition in the fragile alveolar region of the lungs. Hence, there is a great need for identification and prediction of material-associated diseases, currently hindered due to the lack of in-depth understanding of causal relationships, in particular between acute exposures and chronic symptoms. By applying advanced microscopies and omics to in vitro and in vivo systems, together with in silico molecular modeling, it is determined herein that the long-lasting response to a single exposure can originate from the interplay between the newly discovered nanomaterial quarantining and nanomaterial cycling between different lung cell types. This new insight finally allows prediction of the spectrum of lung inflammation associated with materials of interest using only in vitro measurements and in silico modeling, potentially relating outcomes to material properties for a large number of materials, and thus boosting safe-by-design-based material development. Because of its profound implications for animal-free predictive toxicology, this work paves the way to a more efficient and hazard-free introduction of numerous new advanced materials into our lives., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

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., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 TiO 2 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 TiO 2 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 TiO 2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO 2 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 TiO 2 tube. Similar histopathological changes were observed for the TiO 2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO 2 NMs indicated that rutile TiO 2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO 2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO 2 NMs are important predictors for the observed pulmonary effects of TiO 2 NMs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Biological functions of cell membranes and their correlation to the heterogeneity of the latter's lipid composition are still poorly understood. Fluorescence provides one of the most versatile tools for studying biological membranes. However, few bright and photostable fluorescent probes for labeling plasma membranes are available. We have designed and synthesized two such probes, 8 and 9, that are based on the thiazole-coumarin scaffold. Both are environment sensitive and exhibit similar shifts of emission spectra in a variety of solvents as probes based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). In particular, the second, positively charged probe 9 labels the plasma membrane selectively with limited redistribution to other membranes of the cell. Unfortunately, compared to the other two probes tested, 8 and 6-NBD-PC, it exhibits the highest rate of photobleaching. Nevertheless, these new thiazole-coumarin based membrane probes provide a viable approach to the design of novel membrane probes.

The potential of live-cell stimulated emission depletion (STED) nanoscopy has not yet been fully exploited. Currently, the main limitation is the small number of fluorophores and probes that can sustain high light intensity/high dose employed in STED. Namely, fluorophores suitable for STED nanoscopy must be bright and highly photostable and exhibit a large Stokes shift. To expand the list of available probes, we synthesized and evaluated several new membrane probes for live-cell STED nanoscopy. Of the tested probes, probes MePyr500, ThiaCN545 and NB640 not only allow high-resolution STED images, but also partition into the intracellular membranes relatively quickly, thus lacking the selectivity of labelling solely the plasma membrane. During experiments, cytotoxicity was observed merely with the probe ThiaCN545, which blebs the plasma membrane. In comparison with commercially available CellMask Orange and STAR RED (KK114) DPPE, all our tested probes exhibited better photostability with the exception of NB640, which had the fastest bleaching rate of all tested probes. The best overall results can be assigned to the probe MePyr500, providing high-resolution STED images as well as high photostability with no noticeable cytotoxicity, making it an excellent candidate for further development.

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.

Bacterial infections acquired in healthcare facilities including hospitals, the so called healthcare acquired or nosocomial infections, are still of great concern worldwide and represent a significant economical burden. One of the major causes of morbidity is infection with Methicillin Resistant Staphylococcus aureus (MRSA), which has been reported to survive on surfaces for several months. Bactericidal activity of copper-TiO2 thin films, which release copper ions and are deposited on glass surfaces and heated to high temperatures, is well known even when illuminated with very weak UVA light of about 10 μW/cm2. Lately, there is an increased intrerest for one-dimensional TiO2 nanomaterials, due to their unique properties, low cost, and high thermal and photochemical stability. Here we show that copper doped TiO2 nanotubes produce about five times more ·OH radicals as compared to undoped TiO2 nanotubes and that effective surface disinfection, determined by a modified ISO 22196:2011 test, can be achieved even at low intensity UVA light of 30 μW/cm2. The nanotubes can be deposited on a preformed surface at room temperature, resulting in a stable deposition resistant to multiple washings. Up to 103 microorganisms per cm2 can be inactivated in 24 hours, including resistant strains such as Methicillin-resistant Staphylococcus aureus (MRSA) and Extended-spectrum beta-lactamase Escherichia coli (E. coli ESBL). This disinfection method could provide a valuable alternative to the current surface disinfection methods., Competing Interests: The authors have declared that no competing interests exist.

High economic burden is associated with foodborne illnesses. Different disinfection methods are therefore employed in food processing industry; such as use of ultraviolet light or usage of surfaces with copper-containing alloys. However, all the disinfection methods currently in use have some shortcomings. In this work we show that copper doped TiO2 nanotubes deposited on existing surfaces and illuminated with ceiling mounted fluorescent lights can retard the growth of Listeria Innocua by 80% in seven hours of exposure to the fluorescent lights at different places in a food processing plant or in the laboratory conditions with daily reinocuation and washing. The disinfection properties of the surfaces seem to depend mainly on the temperature difference of the surface and the dew point, where for the maximum effectiveness the difference should be about 3 degrees celsius. The TiO2 nanotubes have a potential to be employed for an economical and continuous disinfection of surfaces.

Generating activatable probes that report about molecular vicinity through contact-based mechanisms such as aggregation can be very convenient. Specifically, such probes change a particular spectral property only at the intended biologically relevant target. Xanthene derivatives, for example rhodamines, are able to form aggregates. It is typical to examine aggregation by absorption spectroscopy but for microscopy applications utilizing fluorescent probes it is very important to perform characterization by measuring fluorescence spectra. First we show that excitation spectra of aqueous solutions of rhodamine 6G can be very informative about the aggregation features. Next we establish the dependence of the fluorescence emission spectral maximum shift on the dimer concentration. The obtained information helped us confirm the possibility of aggregation of a recently designed and synthesized rhodamine 6G-based pH-activatable fluorescent probe and to study its pH and concentration dependence. The size of the aggregation-induced emission spectral shift at specific position on the sample can be measured by fluorescence microspectroscopy, which at particular pH allows estimation of the local concentration of the observed probe at microscopic level. Therefore, we show that besides aggregation-caused quenching and aggregation-induced emission also aggregation-induced emission spectral shift can be a useful photophysical phenomenon., (Copyright © 2017 Elsevier B.V. All rights reserved.)

DC-SIGN, an antigen-uptake receptor in dendritic cells (DCs), has a clear role in the immune response but, conversely, can also facilitate infection by providing entry of pathogens into DCs. The key action in both processes is internalization into acidic endosomes and lysosomes. Molecular probes that bind to DC-SIGN could thus provide a useful tool to study internalization and constitute potential antagonists against pathogens. So far, only large molecules have been used to directly observe DC-SIGN-mediated internalization into DCs by fluorescence visualization. We designed and synthesized an appropriate small glycomimetic probe. Two particular properties of the probe were exploited: activation in a low-pH environment and an aggregation-induced spectral shift. Our results indicate that small glycomimetic molecules could compete with antigen/pathogen for binding not only outside but also inside the DC, thus preventing the harmful action of pathogens that are able to intrude into DCs, for example, HIV-1., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Understanding the cell-biomaterial interface from the very first contact is of crucial importance for their successful implementation and function in damaged tissues. However, the lack of bio- and mechano-analytical methods to investigate and probe the initial processes on the interface, especially in 3D, raises the need for applying new experimental techniques. In our study, optical tweezers combined with confocal fluorescence microscopy were optimized to investigate the initial cell-scaffold contact and to investigate its correlation with the material-dependent cell growth. By the optical tweezers-induced cell manipulation accompanied by force detection up to 100 pN and position detection by fluorescence microscopy, accurate adhesion dynamics and strength analysis was implemented, where several attachment sites were formed on the interface in the first few seconds. More importantly, we have shown that dynamics of cell adhesion on scaffold surfaces correlates with cell growth on the days scale, which indicates that the first seconds of the contact could markedly direct further cell response. Such a contact dynamics analysis on 3D scaffold surfaces, applied for the first time, can thus serve to predict scaffold biocompatibility.

Background: The development of efficient drug delivery systems to transport therapeutics across barrier-forming cells remains a challenge. Recently it was shown that liposomes containing perifosine, a synthetic analog of lysophosphatidylcholine, efficiently deliver liposome encapsulated content across barrier-forming cells., Methods: To elucidate the mechanism of the delivery, fluorescent and spin labeled analog of perifosine were synthesized and their transport from liposomes to the barrier-forming MDKC cells was measured., Results & Conclusion: Perifosine analogs are rapidly transported from liposomes into cell membranes. The total amount of perifosine accumulated in plasma membranes seems to be the most important factor in efficient transepithelial transport of liposome-encapsulated substances. Lysolipid-containing liposomal formulations seem to be promising candidates as drug delivery systems in general.

This article present new insights in complex relation between surface- and interface-related physicochemical properties and microstructuring of three-dimensional (3D) gelatin scaffolds, being fabricated by simultaneous temperature-controlled freeze-thawing cycle and in situ cross-linking using variable conditions (pH) and molarity of carbodiimide reagents, on the seeding and growth of fibroblast cells with subsequent tracking of their spreading and morphology. Rarely populated cells with rounded morphology and small elongations are observed on negative charge-rich scaffold surface with a lower cross-linking degree (CD), and consequently higher molecular mobility and availability of cell-recognition sequences, in comparison with the prominently elongated and densely populated cells on a positively charged scaffold's surface with higher CD and low mobility. Surface microstructure effect was demonstrated by cell vacuolization and their pure intercommunication being present on scaffold's bottom side with smaller pores (25 ± 19 µm) and pore wall thickness (9 ± 5 µm), over the air-exposed side with twice bigger pores (56 ± 38 µm) and pore wall thicknesses (12 ± 6 µm). Strong correlations of CD (r(2) = 0.96) and local molecular mobility (r(2)  = -0.44) with pH and reagents molarity, as well as microstructure features being related to temperature gradient, imply on possibility to modulate scaffold's properties in a direction to guide cell viability and most likely its genotype development., (© 2014 Wiley Periodicals, Inc.)

Conformational entropy (SΩ) has long been used to theoretically characterize the dynamics of proteins, DNA, and other polymers. Though recent advances enabled its calculation also from simulations and nuclear magnetic resonance (NMR) relaxation experiments, correlated molecular motion has hitherto greatly hindered both numerical and experimental determination, requiring demanding empirical and computational calibrations. Herein, we show that these motional correlations can be estimated directly from the temperature-dependent SΩ series that reveal effective persistence lengths of the polymers, which we demonstrate by measuring SΩ of amphiphilic molecules in model lipid systems by spin-labeling electron paramagnetic resonance (EPR) spectroscopy. We validate our correlation-corrected SΩ meter against the basic biophysical interactions underlying biomembrane formation and stability, against the changes in enthalpy and diffusion coefficients upon phase transitions, and against the energetics of fatty acid dissociation. As the method can be directly applied to conformational analysis of proteins and other polymers, as well as adapted to NMR or polarized fluorescence techniques, we believe that the approach can greatly enrich the scope of experimentally available statistical thermodynamics, offering new physical insights into the behavior of biomolecules.

All atom molecular dynamics (MD) models provide valuable insight into the dynamics of biophysical systems, but are limited in size or length by the high computational demands. The latter can be reduced by simulating long term diffusive dynamics (also known as Langevin dynamics or Brownian motion) of the most interesting and important user-defined parts of the studied system, termed collective variables (colvars). A few hundred nanosecond-long biased MD trajectory can therefore be extended to millisecond lengths in the colvars subspace at a very small additional computational cost. In this work, we develop a method for determining multidimensional anisotropic position- and timescale-dependent diffusion coefficients (D) by analysing the changes of colvars in an existing MD trajectory. As a test case, we obtained D for dihedral angles of the alanine dipeptide. An open source Mathematica(®) package, capable of determining and visualizing D in one or two dimensions, is available at https://github.com/lbf-ijs/DiffusiveDynamics. Given known free energy and D, the package can also generate diffusive trajectories.

The interactions of tricyclic antidepressant drug, clomipramine (CLO), with pig ear stratum corneum (SC) and model membranes were investigated by electron paramagnetic resonance (EPR) spin labeling to get some insight into the possible application of this drug in transdermal delivery. The changes in membrane characteristics caused by CLO in the regions that are close to the water-lipid interfaces and the central parts of the membranes were searched. The experimental results were supported by computer simulation of EPR spectra, which showed heterogeneity of the membranes composed of regions with different fluidity characteristics. CLO was effective in both parts of the layers, indicating intercalation of the drug into model membranes as well as into the pig ear SC. Introduction of various molar ratios of CLO caused a decrease in the order parameter and an increase in the rotational diffusion of nitroxide moiety in different membrane regions as well as an increase in the polarity of spin probe environment. It also changed the number of resolved spectral components, which reflects the heterogeneity of the membrane. The fluidizing effect of CLO on pig ear SC throughout the whole membrane layers indicates that CLO penetrates into the SC, which is important for its transdermal delivery., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)

A serious drawback of ESR, particularly in its application to cells, is the lack of information on the location of spin probes in the system. In order to realize real time tracking, a spin probe was combined with a fluorophore in a new kind of nitroxide-fluorophore double probe which, in addition to information about lipid dynamics, enables visualization by fluorescence microscopy. The two sets of probes synthesized are based on an amino-alkyne-functionalized sugar that serves as a central polar group and as a linker between the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore and the derivative of the spin labelled fatty acid. In this setting, the location of the fluorophore is restricted to the water-lipid interface, while the nitroxide is located deep in the lipid bilayer. Preliminary tests on cells show preferential localization of both probes in the plasma membrane, with a relatively slow redistribution to other membranes of the cell. We believe that such double probes would be particularly useful for studies of plasma membrane heterogeneity and associated cellular processes.