1,241 research outputs found

    Effect of silver content on the structure and antibacterial activity of silver-doped phosphate-based glasses

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    Staphylococcus aureus can cause a range of diseases, such as osteomyelitis, as well as colonize implanted medical devices. In most instances the organism forms biofilms that not only are resistant to the body's defense mechanisms but also display decreased susceptibilities to antibiotics. In the present study, we have examined the effect of increasing silver contents in phosphate-based glasses to prevent the formation of S. aureus biofilms. Silver was found to be an effective bactericidal agent against S. aureus biofilms, and the rate of silver ion release (0.42 to 1.22 µg·mm–2·h–1) from phosphate-based glass was found to account for the variation in its bactericidal effect. Analysis of biofilms by confocal microscopy indicated that they consisted of an upper layer of viable bacteria together with a layer (20 µm) of nonviable cells on the glass surface. Our results showed that regardless of the silver contents in these glasses (10, 15, or 20 mol%) the silver exists in its +1 oxidation state, which is known to be a highly effective bactericidal agent compared to that of silver in other oxidation states (+2 or +3). Analysis of the glasses by 31P nuclear magnetic resonance imaging and high-energy X-ray diffraction showed that it is the structural rearrangement of the phosphate network that is responsible for the variation in silver ion release and the associated bactericidal effectiveness. Thus, an understanding of the glass structure is important in interpreting the in vitro data and also has important clinical implications for the potential use of the phosphate-based glasses in orthopedic applications to deliver silver ions to combat S. aureus biofilm infections

    Using magnets and magnetic beads to dissect signaling pathways activated by mechanical tension applied to cells

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    Cellular tension has implications in normal biology and pathology. Membrane adhesion receptors serve as conduits for mechanotransduction that lead to cellular responses. Ligand-conjugated magnetic beads are a useful tool in the study of how cells sense and respond to tension. Here we detail methods for their use in applying tension to cells and strategies for analyzing the results. We demonstrate the methods by analyzing mechanotransduction through VE-cadherin on endothelial cells using both permanent magnets and magnetic tweezers

    Label-free biomarkers of human embryonic stem cell differentiation to hepatocytes

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    Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting

    Influence of Paclitaxel Nanomedicine on the Pancreatic Tumor Immune Components

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    Pancreatic cancer (PanCa) is one of the leading causes of cancer-related mortalities in the U.S due to ineffective therapeutic options. Pancreatic tumors are highly desmoplastic and inhibit efficient uptake of therapeutic payloads. Paclitaxel (PTX) has been tested in PanCa therapy with marginally better clinical outcomes, but remain limited by its poor hemocompatibility, biodistribution and intracellular accumulation in tumor cells. Thus, we synthesized a next generation nanoparticle system for PTX to improve its pharmacokinetics and pharmacodynamics (PKPD) in treating PanCa. We also examined ability of the nano formulation to potentiate gemcitabine (GEM) activity in combating chemoresistance in the pancreatic tumor microenvironment (TME).We generated a multi-layered Pluronic F127 coated paclitaxel loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PPNPs) which demonstrated superior therapeutic efficacy over free PTX, effectively potentiated GEM activity in the pancreatic TME, and efficiently reprogrammed oncogenic M2 macrophages to an M1 profile to maintain a pro-inflammatory phenotype

    Investigating the Effect of Surface Properties on Ice Scaling in Eutectic Freeze Crystallization

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    Eutectic Freeze Crystallization (EFC) is an innovative technology that can be applied to treat reverse osmosis (RO) waste streams (brines), to produce pure salt and water. Scaling of the heat exchanger (HX) surface by both ice and salt is currently one of the major drawbacks in the industrial implementation of EFC. At present scaling is controlled by the use of mechanical scraping, which is susceptible to mechanical breakdown, thus reducing the overall process efficiency. Previous studies have shown that lower surface energy materials delay the onset of freezing, and that smooth surfaces reduce nucleation and adhesion sites, thereby reducing the probability of scale formation. Therefore, this study aimed to investigate how the HX surface properties affect ice scaling in EFC, without the influence of mechanical scraping. Copper, Aluminium, Stainless Steel 316 and Brass were the selected HX materials. Ice scaling on the HX materials was investigated using a near eutectic 4 wt.% Na2SO4 aqueous solution, in a crystallization test cell uniquely designed to mimic the region near the HX wall of a crystallizer. The Differential Interference Contrast (DIC) technique was used to study the formation of the initial ice scale layer on the HX material used in the test cell. This method of observation was effective, asfor the first time in a continuous system, the crystallization of the initial ice scale layer was observable in-situ and in real-time. Therefore, with this method, it was possible to investigate the evolution of the predominantscaling modes(nucleation and growth), which differed for the different HX surfaces. The difference was proposed to be due to their distinct surface free energies and surface topographies. The effect of surface free energy and topography on the scaling induction time was investigated while operating at similar heat fluxes (similar cooling rates) for all the metals. The scaling induction time decreased with an increase in the surface free energy, with the Aluminium as an outlier. The recorded scaling induction times for Brass, primary-SS316 and Copper were 92.54, 70.95 and 54.06 min, respectively. Aluminium recorded the longestscaling induction time of 134.74 min. Both the polytetrafluoroethylene (PTFE) coated-SS316 and the primary-SS316 HX surface were used to investigate further the effect of surface free energy on the scaling induction time. The PTFE-coated-SS316 was found to increase the scaling induction times 2.79-fold at a coolant temperature of -15°C, compared to that of the primary-SS316. However, at -20°C and -25°C, the scaling induction times on both surfaces were comparable, which indicated that the benefit of using a low surface free energy material was limited by the cooling rate of the system. It was also found that the scaling induction times were shorter when using a rough-SS316 HX plate, compared to the primary-SS316, because of the larger surface area available for heat transfer. The end of the scaling induction time was characterised by the heterogeneous nucleation and subsequent growth of the ice on the HX surfaces. There was no direct correlation between the HX surface free energy and the nucleation and growth rates. This was because the Brass, Aluminium, SS316 and Copper plates all consist of different surface topographies which also influenced the nucleation and growth rates. However, the nucleation rates consistently increased when the scaling induction times were longer, regardless of the HX material used. The presence of deep sharp crevices on the primary-SS316 also enhanced nucleation rates. These deep sharp crevices created regions of high local supersaturation, where heterogenous nucleation predominated. It was, therefore, reasonable to conclude that the ice scaling induction time was increased by using smooth materials and those of lower surface free energy. The scaling mode was dependent on the surface topography and length of the ice scaling induction time, as longer ice scaling induction times resulted in heterogenous nucleation dominated scaling mode and vice versa. Materials that had a low surface free energy and were smooth minimised the nucleation rate, resulting in a reduced overall scaling rate

    Nanoparticle Induced Cell Magneto-Rotation for the Multiplexed Monitoring of Morphology, Stress and Drug Sensitivity of Suspended Single Cancer Cells.

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    The metastatic process of a cancer relies on the transformation of some of the primary tumor cells into cells capable of migrating through the Extra-Cellular Matrix (ECM), surrounding the tumor, into the bloodstream and the lymph nodes, and then settle in distant tissue, growing new secondary tumors. By identifying, characterizing and quantifying these cells, the progression of cancer in a patient during therapy can be more accurately assessed. Here we describe the development of a new method for quantitative real time monitoring of cell size and morphology, on single live suspended cancer cells, unconfined in three dimensions. The enabling cell magnetorotation (CM) method is made possible by nanoparticle induced cell magnetization. Using a rotating magnetic field, the magnetically labeled cells are actively rotated, then imaged, using a high definition CCD camera. Under proper conditions, the rotation period of a magnetic object is proportional to its shape factor. We demonstrate first that the rotational period, when measured in real-time, can serve to track cellular response to drugs, cytotoxic agents and other chemical stimuli. In addition, while cells are rotated, they exhibit very specific morphological activities, even without a chemical stimulus. Described also is how to multiplex the CM method, to image several dozens to several thousands of cells simultaneously, and using morphology to classify cells into different phenotypic categories, with each phenotype being correlated with malignancy level. The intrinsic tumor heterogeneity, at the cellular level, can be visualized with relationship graphs. Shown is the ability to monitor cell morphological changes over long periods of time, in real time, in order to detect the metastatic potential for heterogeneous populations of cancer cells, using tools from statistical analysis methods. The method relies on unsupervised Machine Learning algorithms which do not require human inputs. Overall it is demonstrated that the CM method can be used as a diagnostic tool to evaluate the phenotypical heterogeneity in a cell population in general, and in a cancer cell population in particular. This fast and high throughput method promises to efficiently assess the efficacy of personalized therapeutic strategies.PHDApplied PhysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111434/1/relbez_1.pd

    Contact X-ray microscopy of living cells by using LiF crystal as imaging detector

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    In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single-shot soft X-ray contact microscopy is presented. High resolved X-ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizingX-ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contactwith a LiF crystal solid-state Xray imaging detector. After exposure and sample removal, the images stored in LiF by the softX-ray contactmicroscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X-ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, thewidedynamicrangeof theLiF imaging detector contributes to obtain high-quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light

    Contractility Dominates Adhesive Ligand Density in Regulating Cellular De-adhesion and Retraction Kinetics

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    Cells that are enzymatically detached from a solid substrate rapidly round up as the tensile prestress in the cytoskeleton is suddenly unopposed by cell–ECM adhesions. We recently showed that this retraction follows sigmoidal kinetics with time constants that correlate closely with cortical stiffness values. This raises the promising prospect that these de-adhesion measurements may be used for high-throughput screening of cell mechanical properties; however, an important limitation to doing so is the possibility that the retraction kinetics may also be influenced and potentially rate-limited by the time needed to sever matrix adhesions. In this study, we address this open question by separating contributions of contractility and adhesion to cellular de-adhesion and retraction kinetics. We first develop serum-free conditions under which U373 MG glioma cells can be cultured on substrates of fixed fibronectin density without direct matrix contributions from the medium. We show that while spreading area increases with ECM protein density, cortical stiffness and the time constants of retraction do not. Conversely, addition of lysophosphatidic acid (LPA) to stimulate cell contractility strongly speeds retraction, independent of the initial matrix protein density and LPA’s contributions to spreading area. All of these trends hold in serum-rich medium commonly used in tissue culture, with the time constants of retraction much more closely tracking cortical stiffness than adhesive ligand density or cell spreading. These results support the use of cellular de-adhesion measurements to track cellular mechanical properties

    A Shift in Central Metabolism Accompanies Virulence Activation in Pseudomonas aeruginosa.

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    The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period.IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach

    Rickettsiae Induce Microvascular Hyperpermeability via Phosphorylation of VE-Cadherins: Evidence from Atomic Force Microscopy and Biochemical Studies

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    The most prominent pathophysiological effect of spotted fever group (SFG) rickettsial infection of microvascular endothelial cells (ECs) is an enhanced vascular permeability, promoting vasogenic cerebral edema and non-cardiogenic pulmonary edema, which are responsible for most of the morbidity and mortality in severe cases. To date, the cellular and molecular mechanisms by which SFG Rickettsia increase EC permeability are largely unknown. In the present study we used atomic force microscopy (AFM) to study the interactive forces between vascular endothelial (VE)-cadherin and human cerebral microvascular EC infected with R. montanensis, which is genetically similar to R. rickettsii and R. conorii, and displays a similar ability to invade cells, but is non-pathogenic and can be experimentally manipulated under Biosafety Level 2 (BSL2) conditions. We found that infected ECs show a significant decrease in VE-cadherin-EC interactions. In addition, we applied immunofluorescent staining, immunoprecipitation phosphorylation assay, and an in vitro endothelial permeability assay to study the biochemical mechanisms that may participate in the enhanced vascular permeability as an underlying pathologic alteration of SFG rickettsial infection. A major finding is that infection of R. montanensis significantly activated tyrosine phosphorylation of VE-cadherin beginning at 48 hr and reaching a peak at 72 hr p.i. In vitro permeability assay showed an enhanced microvascular permeability at 72 hr p.i. On the other hand, AFM experiments showed a dramatic reduction in VE-cadherin-EC interactive forces at 48 hr p.i. We conclude that upon infection by SFG rickettsiae, phosphorylation of VE-cadherin directly attenuates homophilic protein–protein interactions at the endothelial adherens junctions, and may lead to endothelial paracellular barrier dysfunction causing microvascular hyperpermeability. These new approaches should prove useful in characterizing the antigenically related SFG rickettsiae R. conorii and R. rickettsii in a BSL3 environment. Future studies may lead to the development of new therapeutic strategies to inhibit the VE-cadherin-associated microvascular hyperpermeability in SFG rickettsioses
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