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- ItemShelf-Life Evaluation and Lyophilization of PBCA-Based Polymeric Microbubbles(Basel : MDPI, 2019) Ojha, Tarun; Pathak, Vertika; Drude, Natascha; Weiler, Marek; Rommel, Dirk; Rütten, Stephan; Geinitz, Bertram; van Steenbergen, Mies J.; Storm, Gert; Kiessling, Fabian; Lammers, TwanPoly(n-butyl cyanoacrylate) microbubbles (PBCA-MB) are extensively employed for functional and molecular ultrasound (US) imaging, as well as for US-mediated drug delivery. To facilitate the use of PBCA-MB as a commercial platform for biomedical applications, it is important to systematically study and improve their stability and shelf-life. In this context, lyophilization (freeze drying) is widely used to increase shelf-life and promote product development. Here, we set out to analyze the stability of standard and rhodamine-B loaded PBCA-MB at three different temperatures (4 °C, 25 °C, and 37 °C), for a period of time of up to 20 weeks. In addition, using sucrose, glucose, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG) as cryoprotectants, we investigated if PBCA-MB can be lyophilized without affecting their size, concentration, US signal generation properties, and dye retention. Stability assessment showed that PBCA-MB remain largely intact for three and four weeks at 4 °C and 25 °C, respectively, while they disintegrate within one to two weeks at 37 °C, thereby compromising their acoustic properties. Lyophilization analyses demonstrated that PBCA-MB can be efficiently freeze-dried with 5% sucrose and 5% PVP, without changing their size, concentration, and US signal generation properties. Experiments involving rhodamine-B loaded MB indicated that significant dye leakage from the polymeric shell takes place within two to four weeks in case of non-lyophilized PBCA-MB. Lyophilization of rhodamine-loaded PBCA-MB with sucrose and PVP showed that the presence of the dye does not affect the efficiency of freeze-drying, and that the dye is efficiently retained upon MB lyophilization. These findings contribute to the development of PBCA-MB as pharmaceutical products for preclinical and clinical applications.
- ItemDecreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress(Washington, DC : Soc., 2019) Liu, Boqun; Hasrat, Zarief; Poolman, Bert; Boersma, Arnold J.; Mullineaux, Conrad W.Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well.IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm.
- ItemhMRI - A toolbox for quantitative MRI in neuroscience and clinical research(Orlando, Fla. : Academic Press, 2019) Tabelow, Karsten; Balteau, Evelyne; Ashburner, John; Callaghan, Martina F.; Draganski, Bogdan; Helms, Gunther; Kherif, Ferath; Leutritz, Tobias; Lutti, Antoine; Phillips, Christophe; Reimer, Enrico; Ruthotto, Lars; Seif, Maryam; Weiskopf, Nikolaus; Ziegler, Gabriel; Mohammadi, SiawooshNeuroscience and clinical researchers are increasingly interested in quantitative magnetic resonance imaging (qMRI) due to its sensitivity to micro-structural properties of brain tissue such as axon, myelin, iron and water concentration. We introduce the hMRI-toolbox, an open-source, easy-to-use tool available on GitHub, for qMRI data handling and processing, presented together with a tutorial and example dataset. This toolbox allows the estimation of high-quality multi-parameter qMRI maps (longitudinal and effective transverse relaxation rates and , proton density and magnetisation transfer saturation) that can be used for quantitative parameter analysis and accurate delineation of subcortical brain structures. The qMRI maps generated by the toolbox are key input parameters for biophysical models designed to estimate tissue microstructure properties such as the MR g-ratio and to derive standard and novel MRI biomarkers. Thus, the current version of the toolbox is a first step towards in vivo histology using MRI (hMRI) and is being extended further in this direction. Embedded in the Statistical Parametric Mapping (SPM) framework, it benefits from the extensive range of established SPM tools for high-accuracy spatial registration and statistical inferences and can be readily combined with existing SPM toolboxes for estimating diffusion MRI parameter maps. From a user's perspective, the hMRI-toolbox is an efficient, robust and simple framework for investigating qMRI data in neuroscience and clinical research.
- ItemTargeting microplastic particles in the void of diluted suspensions(Amsterdam [u.a.] : Elsevier Science, 2019) Islam, Shohana; Apitius, Lina; Jakob, Felix; Schwaneberg, UlrichAccumulation of microplastic in the environment and food chain will be a grand challenge for our society. Polyurethanes are widely used synthetic polymers in medical (e.g. catheters) and industrial products (especially as foams). Polyurethane is not abundant in nature and only a few microbial strains (fungi and bacteria) and enzymes (polyurethaneases and cutinases) have been reported to efficiently degrade polyurethane. Notably, in nature a long period of time (from 50 to >100 years depending on the literature) is required for degradation of plastics. Material binding peptides (e.g. anchor peptides) bind strongly to polymers such as polypropylene, polyethylene terephthalate, and polyurethane and can target specifically polymers. In this study we report the fusion of the anchor peptide Tachystatin A2 to the bacterial cutinase Tcur1278 which accelerated the degradation of polyester-polyurethane nanoparticles by a factor of 6.6 in comparison to wild-type Tcur1278. Additionally, degradation half-lives of polyester-polyurethane nanoparticles were reduced from 41.8 h to 6.2 h (6.7-fold) in a diluted polyester-polyurethane suspension (0.04% w/v).
- ItemDetailed Fluid Inclusion and Stable Isotope Analysis on Deep Carbonates from the North Alpine Foreland Basin to Constrain Paleofluid Evolution(London : Hindawi, 2019) Mraz, Elena; Wolfgramm, Markus; Moeck, Inga; Thuro, KuroschThe recent interest on environmentally friendly energy resources has increased the economic interest on the Upper Jurassic carbonate rocks in the North Alpine Foreland Basin, which serves as a hydrogeothermal reservoir. An economic reservoir use by geothermal fluid extraction and injection requires a decent understanding of porosity–permeability evolution of the deep laying Upper Jurassic strata at depths greater than 2000 m. The analysis of paleofluids caught in cements of the rock mass helps to determine the postdepositional reservoir evolution and fluid migration. Therefore, the high- and low-permeability areas of the Upper Jurassic in the North Alpine Foreland Basin referred to as Molasse Basin were analyzed by means of encountered postdepositional cements to determine the reservoir evolution. The cements were sampled at different hydrocarbon and geothermal wells, as well as at outcrops in the Franconian and Swabian Alb. To determine the composition and temperature of the paleofluids, fluid inclusions and cements of the Upper Jurassic carbonate rocks were analyzed by microthermometry and stable isotope measurements. Since drill cuttings are a rather available sample material compared to drill cores, a new microthermometry measurement method was achieved for the around 1 mm drill cuttings. Salinity and formation temperature of paleofluids in fluid inclusions and isotope data are consistent with previous studies and reveal a 5-stage evolution: the main cementation phases are composed of (I) the early diagenesis in limestones (200-400 m, 40-50°C), (II) early diagenetic dolomitization, and (III) burial dolomitization (1-2 km, II: 40-90°C; III: 70-100°C; 40 g/L NaCl equiv.), and (IV) late burial calcification (IIIa: 110-140°C, IIIb: 140-200°C) linked to tectonic features in the Molasse Basin. In the outcrop samples, a subsequent (V) cementation phase was determined controlled by karstification. In the southwest, an increase in salinity of the fluid inclusions in vein calcites, above the salinity of the Jurassic seawater, highlights the influence of basin fluids (diagenetic, evaporitic). In the other eastern wells, vein calcites have precipitated from a low saline fluid of around 10-20 g/L NaCl equiv. The low salinity and the isotope values support the theory of a continuous influence of descending meteoric fluids. Consequently, the Upper Jurassic seawater has been diluted by a meteoric fluid to a low saline fluid (<1 g/L), especially in areas with high permeability. Here, we show how a better understanding of cementation trajectory at depth can help to generate a better understanding of geothermal usability in deep carbonate reservoirs.
- ItemColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data(San Francisco, California, US : PLOS, 2019) Smit, Jochem H.; Li, Yichen; Warszawik, Eliza M.; Herrmann, Andreas; Cordes, Thorben; Gilestro, Giorgio FSingle-molecule fluorescence microscopy studies of bacteria provide unique insights into the mechanisms of cellular processes and protein machineries in ways that are unrivalled by any other technique. With the cost of microscopes dropping and the availability of fully automated microscopes, the volume of microscopy data produced has increased tremendously. These developments have moved the bottleneck of throughput from image acquisition and sample preparation to data analysis. Furthermore, requirements for analysis procedures have become more stringent given the demand of various journals to make data and analysis procedures available. To address these issues we have developed a new data analysis package for analysis of fluorescence microscopy data from rod-like cells. Our software ColiCoords structures microscopy data at the single-cell level and implements a coordinate system describing each cell. This allows for the transformation of Cartesian coordinates from transmission light and fluorescence images and single-molecule localization microscopy (SMLM) data to cellular coordinates. Using this transformation, many cells can be combined to increase the statistical power of fluorescence microscopy datasets of any kind. ColiCoords is open source, implemented in the programming language Python, and is extensively documented. This allows for modifications for specific needs or to inspect and publish data analysis procedures. By providing a format that allows for easy sharing of code and associated data, we intend to promote open and reproducible research. The source code and documentation can be found via the project’s GitHub page.
- ItemNanocarriers in photodynamic therapy—in vitro and in vivo studies(Malden, MA : Wiley-Blackwell, 2019) Sztandera, Krzysztof; Gorzkiewicz, Michał; Klajnert‐Maculewicz, BarbaraPhotodynamic therapy (PDT) is a minimally invasive technique which has proven to be successful in the treatment of several types of tumors. This relatively simple method exploits three inseparable elements: phototoxic compound (photosensitizer [PS]), light source, and oxygen. Upon irradiation by light with specified wavelength, PS generates reactive oxygen species, which starts the cascade of reactions leading to cell death. The positive therapeutic outcome of PDT may be limited due to several aspects, including low water solubility of PSs, hampering their effective administration and blood circulation, as well as low tumor specificity, inefficient cellular uptake and activation energies requiring prolonged illumination times. One of the promising approaches to overcome these obstacles involves the use of carrier systems modulating pharmacokinetics and pharmacodynamics of the PSs. In the present review, we summarized current in vitro and in vivo studies regarding the use of nanoparticles as potential delivery devices for PSs to enhance their cellular uptake and cytotoxic properties, and thus—the therapeutic outcome of PDT.
- ItemRespiratory tract deposition of inhaled roadside ultrafine refractory particles in a polluted megacity of South-East Asia(Amsterdam [u.a.] : Elsevier Science, 2019) Kecorius, Simonas; Madueño, Leizel; Löndahl, Jakob; Vallar, Edgar; Galvez, Maria Cecilia; Idolor, Luisito F.; Gonzaga-Cayetano, Mylene; Müller, Thomas; Birmili, Wolfram; Wiedensohler, AlfredRecent studies demonstrate that Black Carbon (BC) pollution in economically developing megacities remain higher than the values, which the World Health Organization considers to be safe. Despite the scientific evidence of the degrees of BC exposure, there is still a lack of understanding on how the severe levels of BC pollution affect human health in these regions. We consider information on the respiratory tract deposition dose (DD) of BC to be essential in understanding the link between personal exposure to air pollutants and corresponding health effects. In this work, we combine data on fine and ultrafine refractory particle number concentrations (BC proxy), and activity patterns to derive the respiratory tract deposited amounts of BC particles for the population of the highly polluted metropolitan area of Manila, Philippines. We calculated the total DD of refractory particles based on three metrics: refractory particle number, surface area, and mass concentrations. The calculated DD of total refractory particle number in Metro Manila was found to be 1.6 to 17 times higher than average values reported from Europe and the U.S. In the case of Manila, ultrafine particles smaller than 100 nm accounted for more than 90% of the total deposited refractory particle dose in terms of particle number. This work is a first attempt to quantitatively evaluate the DD of refractory particles and raise awareness in assessing pollution-related health effects in developing megacities. We demonstrate that the majority of the population may be highly affected by BC pollution, which is known to have negative health outcomes if no actions are taken to mitigate its emission. For the governments of such metropolitan areas, we suggest to revise currently existing environmental legislation, raise public awareness, and to establish supplementary monitoring of black carbon in parallel to already existing PM 10 and PM 2.5 measures. © 2019
- ItemNrf2 signaling and inflammation are key events in physical plasma-spurred wound healing(Wyoming, NSW : Ivyspring, 2019) Schmidt, Anke; Woedtke, Thomas, von; Vollmar, Brigitte; Hasse, Sybille; Bekeschus, SanderWound healing is strongly associated with the presence of a balanced content of reactive species in which oxygen-dependent, redox-sensitive signaling represents an essential step in the healing cascade. Numerous studies have demonstrated that cold physical plasma supports wound healing due to its ability to deliver a beneficial mixture of reactive species directly to the cells. Methods: We described a preclinical proof-of-principle-concept of cold plasma use in a dermal, full-thickness wound model in immunocompetent SKH1 mice. Quantitative PCR, Western blot analysis, immunohistochemistry and immunofluorescence were perfomed to evaluate the expression and cellular translocation of essential targets of Nrf2 and p53 signaling as well as immunomodulatory and angiogenetic factors. Apoptosis and proliferation were detected using TUNEL assay and Ki67 staining, respectively. Cytokine levels in serum were measured using bead-based multiplex cytokine analysis. Epidermal keratinocytes and dermal fibroblasts were isolated from mouse skin to perform functional knockdown experiments. Intravital fluorescence analysis was used to illustrate and quantified microvascular features. Results: Plasma exerted significant effects on wound healing in mice, including the promotion of granulation and reepithelialization as a consequence of the migration of skin cells, the balance of antioxidant and inflammatory response, and the early induction of macrophage and neutrophil recruitment to the wound sites. Moreover, through an early and local plasma-induced p53 inhibition with a concomitant stimulation of proliferation, the upregulation of angiogenetic factors, and an increased outgrowth of new vessels, our findings explain why dermal skin repair is accelerated. The cellular redox homeostasis was maintained and cells were defended from damage by a strong modulation of the nuclear E2-related factor (Nrf2) pathway and redox-sensitive p53 signaling. Conclusions: Although acute wound healing is non-problematic, the pathways highlighted that mainly the activation of Nrf2 signaling is a promising strategy for the clinical use of cold plasma in chronic wound healing.
- ItemIsolation of phytochemical constituents from hunteria umbellata k. Schum(Benin : University of Benin, 2019) Ali, Iftikhar; Falodun, Abiodun; Siyo, Baraa; Jules, Bankeu; Hussain, Hidayat; Langer, PeterHunteria umbellata K. Schum has been reported for the treatment of diabetes in Nigeria. In the present study, the ethanolic extract of dried leaves of Hunteria umbellata K. Schum was investigated for the chemical principles. The isolated pure compounds were characterized by NMR, IR and Mass spectral studies. Ursolic acid (1), oleanolic acid (2) and squalene (3) were the main constituents isolated from the extract.