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Sample records for afm fluid cell

  1. Force-controlled manipulation of single cells: from AFM to FluidFM.

    PubMed

    Guillaume-Gentil, Orane; Potthoff, Eva; Ossola, Dario; Franz, Clemens M; Zambelli, Tomaso; Vorholt, Julia A

    2014-07-01

    The ability to perturb individual cells and to obtain information at the single-cell level is of central importance for addressing numerous biological questions. Atomic force microscopy (AFM) offers great potential for this prospering field. Traditionally used as an imaging tool, more recent developments have extended the variety of cell-manipulation protocols. Fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures. The crucial element of the technology is the connection of the hollow cantilevers to a pressure controller, allowing their operation in liquid as force-controlled nanopipettes under optical control. Proof-of-concept studies demonstrated a broad spectrum of single-cell applications including isolation, deposition, adhesion and injection in a range of biological systems. PMID:24856959

  2. AFM indentation study of breast cancer cells

    SciTech Connect

    Li, Q.S.; Lee, G.Y.H.; Ong, C.N.; Lim, C.T.

    2008-10-03

    Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Young's modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 deg. C), and their apparent Young's modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis.

  3. Characterizing Cell Mechanics with AFM and Microfluidics

    NASA Astrophysics Data System (ADS)

    Walter, N.; Micoulet, A.; Suresh, S.; Spatz, J. P.

    2007-03-01

    Cell mechanical properties and functionality are mainly determined by the cytoskeleton, besides the cell membrane, the nucleus and the cytosol, and depend on various parameters e.g. surface chemistry and rigidity, surface area and time available for cell spreading, nutrients and drugs provided in the culture medium. Human epithelial pancreatic and mammary cancer cells and their keratin intermediate filaments are the main focus of our work. We use Atomic Force Microscopy (AFM) to study cells adhering to substrates and Microfluidic Channels to probe cells in suspension, respectively. Local and global properties are extracted by varying AFM probe tip size and the available adhesion area for cells. Depth-sensing, instrumented indentation tests with AFM show a clear difference in contact stiffness for cells that are spread of controlled substrates and those that are loosely attached. Microfluidic Channels are utilized in parallel to evaluate cell deformation and ``flow resistance'', which are dependent on channel cross section, flow rate, cell nucleus size and the mechanical properties of cytoskeleton and membrane. The results from the study are used to provide some broad and quantitative assessments of the connections between cellular/subcellular mechanics and biochemical origins of disease states.

  4. A review of design concepts for the Advanced Fluids Module (AFM) project

    NASA Technical Reports Server (NTRS)

    Hill, Myron E.; Tschen, Peter S.

    1993-01-01

    This paper reviews preliminary fluid module design concepts for the Advanced Fluids Module (AFM) project. The objective of this effort is to provide a facility that can handle a wide variety of fluids experiments. Sample science requirements were written and conceptual designs were subsequently generated during the last year. Experiments from the following fluid physics subject areas were used as conceptual design drivers: static and dynamic interfacial phenomena; bubble/droplet thermocapillary migration; surface tension convection and instabilities; thermal/solutal convection; pool boiling; and multiphase flow. After the conceptual designs were completed, the next phase attempted to combine experiments capabilities into a multipurpose, multiuser apparatus configured for the Space Station Freedom. It was found that all the fluid subject areas considered could be accommodated by three basic types of fluids modules. These modules are the Static Fluid Cell Module, the Dynamic Fluid Cell Module, and the Multiphase Flow Module. Descriptions of these preliminary modules designs and their particular sub-systems (e.g., fluid and thermal systems) are discussed. These designs will be refined as the nature of the flight program becomes clearer over the next six to twelve months.

  5. Surface Morphological Studies on Nerve Cells by AFM

    NASA Astrophysics Data System (ADS)

    Durkaya, Goksel; Zhong, Lei; Rehder, Vincent; Dietz, Nikolaus

    2009-03-01

    Surface morphological properties of fixed and living nerve cells removed from the buccal ganglion of Helisoma trivolvis have been studied by using Atomic Force Microscopy (AFM). Identified, individual neurons were removed from the buccal ganglion of Helisoma trivolvis and plated into poly-L-lysine coated glass cover-slips. The growth of the nerve cells was stopped and fixed with 0.1% Glutaraldehyde and 4% Formaldehyde solution after extension of growth cones at the tip of the axons. Topography and softness of growth cone filopodia and overlying lamellopodium (veil) were probed by AFM. Information obtained from AFM's amplitude and phase channels have been used for determination of softness of the region probed. The results of structural studies on the cells are linked to their mechanical properties and internal molecular density distribution.

  6. Quantitative nano-mechanics of biological cells with AFM

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor

    2013-03-01

    The importance of study of living cells is hard to overestimate. Cell mechanics is a relatively young, yet not a well-developed area. Besides just a fundamental interest, large practical need has emerged to measure cell mechanics quantitatively. Recent studies revealed a significant correlation between stiffness of biological cells and various human diseases, such as cancer, malaria, arthritis, and even aging. However, really quantitative studies of mechanics of biological cells are virtually absent. It is not even clear if the cell, being a complex and heterogeneous object, can be described by the elastic modulus at all. Atomic force microscopy (AFM) is a natural instrument to study properties of cells in their native environments. Here we will demonstrate that quantitative measurements of elastic modulus of cells with AFM are possible. Specifically, we will show that the ``cell body'' (cell without ``brush'' surface layer, a non-elastic layer surrounding cells) typically demonstrates the response of a homogeneous elastic medium up to the deformation of 10-20%, but if and only if a) the cellular brush layer is taken into account, b) rather dull AFM probes are used. This will be justified with the help of the strong condition of elastic behavior of material: the elastic modulus is shown to be independent on the indentation depth. We will also demonstrate that an attempt either to ignore the brush layer or to use sharp AFM probes will result in the violation of the strong condition, which implies impossibility to use the concept of the elastic modulus to describe cell mechanics in such experiments. Examples of quantitative measurements of the Young's modulus of the cell body and the cell brush parameters will be given for various cells. Address when submitting: Clarkson University, Potsdam, NY 13699

  7. Wedged AFM-cantilevers for parallel plate cell mechanics.

    PubMed

    Stewart, Martin P; Hodel, Adrian W; Spielhofer, Andreas; Cattin, Cedric J; Müller, Daniel J; Helenius, Jonne

    2013-04-01

    The combination of atomic force microscopy (AFM) and optical microscopy has gained popularity for mechanical analysis of living cells. In particular, recent AFM-based assays featuring tipless cantilevers and whole-cell deformation have yielded insights into cellular function, structure, and dynamics. However, in these assays the standard ≈10° tilt of the cantilever prevents uniaxial loading, which complicates assessment of cellular geometry and can cause cell sliding or loss of loosely adherent cells. Here, we describe an approach to modify tipless cantilevers with wedges and, thereby, achieve proper parallel plate mechanics. We provide guidance on material selection, the wedge production process, property and geometry assessment, and the calibration of wedged cantilevers. Furthermore, we demonstrate their ability to simplify the assessment of cell shape, prevent lateral displacement of round cells during compression, and improve the assessment of cell mechanical properties. PMID:23473778

  8. Nano-Bio-Mechanics of Neuroblastoma Cells Using AFM

    NASA Astrophysics Data System (ADS)

    Bastatas, Lyndon; Matthews, James; Kang, Min; Park, Soyeun

    2011-10-01

    We have conducted an in vitro study to determine the elastic moduli of neurobalstoma cell lines using atomic force microscopy. Using a panel of cell lines established from neuroblastoma patients at different stages of disease progress and treatment, we have investigated the differences in elastic moduli during a course of cancer progression and chemotherapy. The cells were grown on the hard substrates that are chemically functionalized to enhance adhesion. We have performed the AFM indentation experiments with different applied forces from the AFM probe. For the purpose of the comparison between cell lines, the indentations were performed only on cell centers. The obtained force-distance curves were analyzed using the Hertz model in order to extract the elastic moduli. We have found that the elastic moduli of human neuroblastoma cells significantly varied during the disease progression. We postulate that the observed difference might be affected by the treatment and chemotherapy.

  9. AFM characterization of nanobubble formation and slip condition in oxygenated and electrokinetically altered fluids.

    PubMed

    Bhushan, Bharat; Pan, Yunlu; Daniels, Stephanie

    2013-02-15

    Nanobubbles are gas-filled features that spontaneously form at the interface of hydrophobic surfaces and aqueous solutions. In this study, an atomic force microscope (AFM) was used to characterize the morphology of nanobubbles formed on hydrophobic polystyrene (PS) and octadecyltrichlorosilane (OTS) films immersed in DI water, saline, saline with oxygen and an electrokinetically altered saline solution produced with Taylor-Couette-Poiseuille flow under elevated oxygen pressure. AFM force spectroscopy was used to evaluate hydrodynamic and electrostatic forces and boundary slip condition in various fluids. The effect of solution, electric field and surface charge on shape, size and density of nanobubbles as well as slip length was quantified and the results and underlying mechanisms are presented in this paper. PMID:23123096

  10. SU-8 hollow cantilevers for AFM cell adhesion studies

    NASA Astrophysics Data System (ADS)

    Martinez, Vincent; Behr, Pascal; Drechsler, Ute; Polesel-Maris, Jérôme; Potthoff, Eva; Vörös, Janos; Zambelli, Tomaso

    2016-05-01

    A novel fabrication method was established to produce flexible, transparent, and robust tipless hollow atomic force microscopy (AFM) cantilevers made entirely from SU-8. Channels of 3 μm thickness and several millimeters length were integrated into 12 μm thick and 40 μm wide cantilevers. Connected to a pressure controller, the devices showed high sealing performance with no leakage up to 6 bars. Changing the cantilever lengths from 100 μm to 500 μm among the same wafer allowed the targeting of various spring constants ranging from 0.5 to 80 N m‑1 within a single fabrication run. These hollow polymeric AFM cantilevers were operated in the optical beam deflection configuration. To demonstrate the performance of the device, single-cell force spectroscopy experiments were performed with a single probe detaching in a serial protocol more than 100 Saccharomyces cerevisiae yeast cells from plain glass and glass coated with polydopamine while measuring adhesion forces in the sub-nanoNewton range. SU-8 now offers a new alternative to conventional silicon-based hollow cantilevers with more flexibility in terms of complex geometric design and surface chemistry modification.

  11. An AFM study of the chlorite-fluid interface. [Atomic Force Microscopy

    SciTech Connect

    Vrdoljak, G.A.; Henderson, G.S.; Fawcett, J.J. . Dept. of Geology)

    1992-01-01

    Chlorite is a ubiquitous mineral in many geologic environments and plays an important role in elemental adsorption and retention in soils. Chlorite has a 2:1 layer structure consisting of two tetrahedral sheets with an octahedral sheet between them (talc-like layer). The 2:1 layer is charge balanced and hydrogen-bonded by an interlayer of MgOH[sub 6] octahedra (brucite-like layer). The nature of chlorite's structure, its ease of imaging, and perfect 001 cleavage, make this mineral an ideal substrate for use in elemental adsorption studies in solution, with the AFM. The 001 cleavage plane of a 2b polytype with composition (Mg[sub 4.4]Fe[sub 0.6]Al[sub 1.0])[(Si[sub 2.9]Al[sub 1.1])]O[sub 10](OH)[sub g] has been imaged in air, water, and oil by atomic force microscopy. Dissolution features are observed in water, showing sub-micron features dissolving in real-time. Atomic resolution of both the talc-like and brucite-like layers has been obtained in air. However, only the tetrahedral sheet of the talc-like layer has been imaged at atomic resolution in oil and water, which may indicate a structural instability of the brucite-like surface in solution. Measurements of the unit-cell dimensions (a and b) for the talc-like layer in the three different media indicate a structural expansion of the mineral surface in solution. The a unit cell dimension expands by 7.4 [+-] 0.1% when in water; conversely, the b dimension varies greatly when in oil ([minus]10% to +20%), relative to air. The effects of these solution media on the structure of chlorite are revealed by characterization with the AFM. This information should prove useful in future studies of adsorption onto layer silicates.

  12. Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

    NASA Astrophysics Data System (ADS)

    Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

    2008-09-01

    Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

  13. Probing the PEDOT:PSS/cell interface with conductive colloidal probe AFM-SECM

    NASA Astrophysics Data System (ADS)

    Knittel, P.; Zhang, H.; Kranz, C.; Wallace, G. G.; Higgins, M. J.

    2016-02-01

    Conductive colloidal probe Atomic Force-Scanning Electrochemical Microscopy (AFM-SECM) is a new approach, which employs electrically insulated AFM probes except for a gold-coated colloid located at the end of the cantilever. Hence, force measurements can be performed while biasing the conductive colloid under physiological conditions. Moreover, such colloids can be modified by electrochemical polymerization resulting, e.g. in conductive polymer-coated spheres, which in addition may be loaded with specific dopants. In contrast to other AFM-based single cell force spectroscopy measurements, these probes allow adhesion measurements at the cell-biomaterial interface on multiple cells in a rapid manner while the properties of the polymer can be changed by applying a bias. In addition, spatially resolved electrochemical information e.g., oxygen reduction can be obtained simultaneously. Conductive colloid AFM-SECM probes modified with poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) are used for single cell force measurements in mouse fibroblasts and single cell interactions are investigated as a function of the applied potential.Conductive colloidal probe Atomic Force-Scanning Electrochemical Microscopy (AFM-SECM) is a new approach, which employs electrically insulated AFM probes except for a gold-coated colloid located at the end of the cantilever. Hence, force measurements can be performed while biasing the conductive colloid under physiological conditions. Moreover, such colloids can be modified by electrochemical polymerization resulting, e.g. in conductive polymer-coated spheres, which in addition may be loaded with specific dopants. In contrast to other AFM-based single cell force spectroscopy measurements, these probes allow adhesion measurements at the cell-biomaterial interface on multiple cells in a rapid manner while the properties of the polymer can be changed by applying a bias. In addition, spatially resolved electrochemical

  14. Cell mechanics as a marker for diseases: Biomedical applications of AFM

    NASA Astrophysics Data System (ADS)

    Rianna, Carmela; Radmacher, Manfred

    2016-08-01

    Many diseases are related to changes in cell mechanics. Atomic Force Microscopy (AFM) is one of the most suitable techniques allowing the investigation of both topography and mechanical properties of adherent cells with high spatial resolution under physiological conditions. Over the years the use of this technique in medical and clinical applications has largely increased, resulting in the notion of cell mechanics as a biomarker to discriminate between different physiological and pathological states of cells. Cell mechanics has proven to be a biophysical fingerprint able discerning between cell phenotypes, unraveling processes in aging or diseases, or even detecting and diagnosing cellular pathologies. We will review in this report some of the works on cell mechanics investigated by AFM with clinical and medical relevance in order to clarify the state of research in this field and to highlight the role of cell mechanics in the study of pathologies, focusing on cancer, blood and cardiovascular diseases.

  15. Single cell transfection using plasmid decorated AFM probes

    SciTech Connect

    Cuerrier, Charles M.; Lebel, Rejean; Grandbois, Michel . E-mail: michel.grandbois@usherbrooke.ca

    2007-04-13

    Eukaryotic cells were individually transfected using commercially available atomic force microscope tips decorated with plasmidic DNA encoding for the fluorescent protein EGFP. In a typical transfection attempt, the tip is forcibly incorporated into the cell thus allowing for the transfer of the genetic material through the cell membrane. A sharp discontinuity, corresponding to the passage of the tip through the cell membrane can be easily detected when monitoring the cellular deformation as a function of the applied force. In order for the transfection to be successful, the tip must reversibly penetrates the membrane without causing disturbance or damage to the cell. Transfection success rate (30%), cell survival, and growth are confirmed by epifluorescence microscopy. This technique provides an alternative tool to the transfection toolbox, allowing the transfection of specific individual cells with minimal disturbance.

  16. Single-Molecule Studies of Integrins by AFM-Based Force Spectroscopy on Living Cells

    NASA Astrophysics Data System (ADS)

    Eibl, Robert H.

    The characterization of cell adhesion between two living cells at the single-molecule level, i.e., between one adhesion receptor and its counter-receptor, appears to be an experimental challenge. Atomic force microscopy (AFM) can be used in its force spectroscopy mode to determine unbinding forces of a single pair of adhesion receptors, even with a living cell as a probe. This chapter provides an overview of AFM force measurements of the integrin family of cell adhesion receptors and their ligands. A focus is given to major integrins expressed on leukocytes, such as lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4). These receptors are crucial for leukocyte trafficking in health and disease. LFA-1 and VLA-1 can be activated within the bloodstream from a low-affinity to a high-affinity receptor by chemokines in order to adhere strongly to the vessel wall before the receptor-bearing leukocytes extravasate. The experimental considerations needed to provide near-physiological conditions for a living cell and to be able to measure adequate forces at the single-molecule level are discussed in detail. AFM technology has been developed into a modern and extremely sensitive tool in biomedical research. It appears now that AFM force spectroscopy could enter, within a few years, medical applications in diagnosis and therapy of cancer and autoimmune diseases.

  17. AFM method to detect differences in adhesion of silica bids to cancer and normal epithelial cells

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor; Iyer, Swaminathan; Gaikwad, Ravi; Woodworth, Craig

    2009-03-01

    To date, the methods of detection of cancer cells have been mostly based on traditional techniques used in biology, such as visual identification of malignant changes, cell growth analysis, specific ligand-receptor labeling, or genetic tests. Despite being well developed, these methods are either insufficiently accurate or require a lengthy complicated analysis. A search for alternative methods for the detection of cancer cells may be a fruitful approach. Here we describe an AFM study that may result in a new method for detection of cancer cells in vitro. Here we use atomic force microscopy (AFM) to study adhesion of single silica beads to malignant and normal cells cultured from human cervix. We found that adhesion depends on the time of contact, and can be statistically different for malignant and normal cells. Using these data, one could develop an optical method of cancer detection based on adhesion of various silica beads.

  18. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    PubMed Central

    2009-01-01

    Background Imaging tools such as scanning electron microscope (SEM) and atomic force microscope (AFM) can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK) 293, human breast cancer (MCF-7) and mouse melanoma (B16F1) cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor protein SMAR1 might be

  19. Pericellular Brush and Mechanics of Guinea Pig Fibroblast Cells Studied with AFM.

    PubMed

    Dokukin, Maxim; Ablaeva, Yulija; Kalaparthi, Vivekanand; Seluanov, Andrei; Gorbunova, Vera; Sokolov, Igor

    2016-07-12

    The atomic force microscopy (AFM) indentation method combined with the brush model can be used to separate the mechanical response of the cell body from deformation of the pericellular layer surrounding biological cells. Although self-consistency of the brush model to derive the elastic modulus of the cell body has been demonstrated, the model ability to characterize the pericellular layer has not been explicitly verified. Here we demonstrate it by using enzymatic removal of hyaluronic content of the pericellular brush for guinea pig fibroblast cells. The effect of this removal is clearly seen in the AFM force-separation curves associated with the pericellular brush layer. We further extend the brush model for brushes larger than the height of the AFM probe, which seems to be the case for fibroblast cells. In addition, we demonstrate that an extension of the brush model (i.e., double-brush model) is capable of detecting the hierarchical structure of the pericellular brush, which, for example, may consist of the pericellular coat and the membrane corrugation (microridges and microvilli). It allows us to quantitatively segregate the large soft polysaccharide pericellular coat from a relatively rigid and dense membrane corrugation layer. This was verified by comparison of the parameters of the membrane corrugation layer derived from the force curves collected on untreated cells (when this corrugation membrane part is hidden inside the pericellular brush layer) and on treated cells after the enzymatic removal of the pericellular coat part (when the corrugations are exposed to the AFM probe). We conclude that the brush model is capable of not only measuring the mechanics of the cell body but also the parameters of the pericellular brush layer, including quantitative characterization of the pericellular layer structure. PMID:27410750

  20. Nano-scale Topographical Studies on the Growth Cones of Nerve Cells using AFM

    NASA Astrophysics Data System (ADS)

    Durkaya, Goksel; Zhong, Lei; Rehder, Vincent; Dietz, Nikolaus

    2009-11-01

    Nerve cells are the fundamental units which are responsible for intercommunication within the nervous system. The neurites, fibrous cable-like extensions for information delivery, of nerve cells are tipped by highly motile sensory structures known as the growth cones which execute important functions; neural construction, decision making and navigation during development and regeneration of the nervous system. The highly dynamic subcomponents of the growth cones are important in neural activity. Atomic Force Microscopy (AFM) is the most powerful microscopy technique which is capable of imaging without conductivity constraint and in liquid media. AFM providing nano-scale topographical information on biological structures is also informative on the physical properties such as: elasticity, adhesion, and softness. This contribution focuses on AFM analysis of the growth cones of the nerve cells removed from the buccal ganglion of Helisoma trivolvis. The results of nano-scale topography and softness analysis on growth cone central domain, filopodia and overlying lamellopodium (veil) are presented. The subcomponents of the growth cones of different nerve cells are compared to each other. The results of the analysis are linked to the mechanical properties and internal molecular density distribution of the growth cones.

  1. An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

    SciTech Connect

    Zhang, Xiaojun; Chen, Yuan; Chen, Yong

    2014-03-28

    Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.

  2. Cell visco-elasticity measured with AFM and optical trapping at sub-micrometer deformations.

    PubMed

    Nawaz, Schanila; Sánchez, Paula; Bodensiek, Kai; Li, Sai; Simons, Mikael; Schaap, Iwan A T

    2012-01-01

    The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30-600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components. PMID:23028915

  3. Simulation of CNT-AFM tip based on finite element analysis for targeted probe of the biological cell

    NASA Astrophysics Data System (ADS)

    Yousefi, Amin Termeh; Mahmood, Mohamad Rusop; Miyake, Mikio; Ikeda, Shoichiro

    2016-07-01

    Carbon nanotubes (CNTs) are potentially ideal tips for atomic force microscopy (AFM) due to the robust mechanical properties, nano scale diameter and also their ability to be functionalized by chemical and biological components at the tip ends. This contribution develops the idea of using CNTs as an AFM tip in computational analysis of the biological cell's. Finite element analysis employed for each section and displacement of the nodes located in the contact area was monitored by using an output database (ODB). This reliable integration of CNT-AFM tip process provides a new class of high performance nanoprobes for single biological cell analysis.

  4. Cellular mechanoadaptation to substrate mechanical properties: contributions of substrate stiffness and thickness to cell stiffness measurements using AFM.

    PubMed

    Vichare, Shirish; Sen, Shamik; Inamdar, Mandar M

    2014-02-28

    Mechanosensing by adherent cells is usually studied by quantifying cell responses on hydrogels that are covalently linked to a rigid substrate. Atomic force microscopy (AFM) represents a convenient way of characterizing the mechanoadaptation response of adherent cells on hydrogels of varying stiffness and thickness. Since AFM measurements reflect the effective cell stiffness, therefore, in addition to measuring real cytoskeletal alterations across different conditions, these measurements might also be influenced by the geometry and physical properties of the substrate itself. To better understand how the physical attributes of the gel influence AFM stiffness measurements of cells, we have used finite element analysis to simulate the indentation of cells of various spreads resting on hydrogels of varying stiffness and thickness. Consistent with experimental results, our simulation results indicate that for well spread cells, stiffness values are significantly over-estimated when experiments are performed on cells cultured on soft and thin gels. Using parametric studies, we have developed scaling relationships between the effective stiffness probed by AFM and the bulk cell stiffness, taking cell and tip geometry, hydrogel properties, nuclear stiffness and cell contractility into account. Finally, using simulated mechanoadaptation responses, we have demonstrated that a cell stiffening response may arise purely due to the substrate properties. Collectively, our results demonstrate the need to take hydrogel properties into account while estimating cell stiffness using AFM indentation. PMID:24651595

  5. Raman confocal microscopy and AFM combined studies of cancerous cells treated with Paclitaxel

    NASA Astrophysics Data System (ADS)

    Derely, L.; Collart Dutilleul, P.-Y.; Michotte de Welle, Sylvain; Szabo, V.; Gergely, C.; Cuisinier, F. J. G.

    2011-03-01

    Paclitaxel interferes with the normal function of microtubule breakdown, induces apoptosis in cancer cells and sequesters free tubulin. As this drug acts also on other cell mechanisms it is important to monitor its accumulation in the cell compartments. The intracellular spreading of the drug was followed using a WITEC 300R confocal Raman microscope equipped with a CCD camera. Hence Atomic force microscopy (an MFP3D- Asylum Research AFM) in imaging and force mode was used to determine the morphological and mechanical modifications induced on living cells. These studies were performed on living epithelial MCF-7 breast cancer cells. Paclitaxel was added to cell culture media for 3, 6 and 9 hours. Among the specific paclitaxel Raman bands we selected the one at 1670 cm-1 because it is not superposed by the spectrum of the cells. Confocal Raman images are formed by monitoring this band, the NH2 and the PO4 band. Paclitaxel slightly accumulates in the nucleus forming patches. The drug is also concentrated in the vicinity of the cell membrane and in an area close to the nucleus where proteins accumulate. Our AFM images reveal that the treated cancerous MCF-7 cells keep the same size as the non treated ones, but their shape becomes more oval. Cell's elasticity is also modified: a difference of 2 kPa in the Young Modulus characterizes the treated MCF-7 mammary cancerous cell. Our observations demonstrate that paclitaxel acts not only on microtubules but accumulates also in other cell compartments (nucleus) where microtubules are absent.

  6. DNA-coated AFM cantilevers for the investigation of cell adhesion and the patterning of live cells

    SciTech Connect

    Hsiao, Sonny C.; Crow, Ailey K.; Lam, Wilbur A.; Bertozzi, Carolyn R.; Fletcher, Daniel A.; Francis, Matthew B.

    2008-08-01

    Measurement of receptor adhesion strength requires the precise manipulation of single cells on a contact surface. To attach live cells to a moveable probe, DNA sequences complementary to strands displayed on the plasma membrane are introduced onto AFM cantilevers (see picture, bp=base pairs). The strength of the resulting linkages can be tuned by varying the length of DNA strands, allowing for controlled transport of the cells.

  7. Measuring cell wall elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM

    SciTech Connect

    Beckmann, Melissa; Venkataraman, Sankar; Doktycz, Mitchel John; Nataro, James P; Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P

    2006-07-01

    Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

  8. Towards quantitative molecular mapping of cells by Raman microscopy: using AFM for decoupling molecular concentration and cell topography.

    PubMed

    Boitor, Radu; Sinjab, Faris; Strohbuecker, Stephanie; Sottile, Virginie; Notingher, Ioan

    2016-06-23

    Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml(-1), while proteins were distributed more uniformly and reached concentrations as high as ∼50 ± 12 mg ml(-1). The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing

  9. Label-free and quantitative evaluation of cytotoxicity based on surface nanostructure and biophysical property of cells utilizing AFM.

    PubMed

    Lee, Young Ju; Lee, Gi-Ja; Kang, Sung Wook; Cheong, Youjin; Park, Hun-Kuk

    2013-06-01

    In this study, the four commonly used cytotoxicity assays and the mechanical properties as evaluated by atomic force microscopy (AFM) were compared in a cellular system. A cytotoxicity assay is the first and most essential test to evaluate biocompatibility of various toxic substances. Many of the cytotoxicity methods require complicated and labor-intensive process, as well as introduce experimental error. In addition, these methods cannot provide instantaneous and quantitative cell viability information. AFM has become an exciting analytical tool in medical, biological, and biophysical research due to its unique abilities. AFM-based force-distance curve measurements precisely measure the changes in the biophysical properties of the cell. Therefore, we observed the morphological changes and mechanical property changes in L929 cells following sodium lauryl sulfate (SLS) treatment utilizing AFM. AFM imaging showed that the toxic effects of SLS changed not only the spindle-like shape of L929 cells into a round shape, but also made a rough cell surface. As the concentration of SLS was increased, the surface roughness of L929 cell was increased, and stiffness decreased. We confirmed that inhibition of proliferation clearly increased with increases in SLS concentration based on results from MTT, WST, neutral red uptake, and LIVE/DEAD viability/cytotoxicity assays. The estimated IC₅₀ value by AFM analysis was similar to those of other conventional assays and was included within the 95% confidence interval range. We suggest that an AFM quantitative analysis of the morphological and biophysical changes in cells can be utilized as a new method for evaluating cytotoxicity. PMID:23582483

  10. Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

    NASA Astrophysics Data System (ADS)

    Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

    2011-11-01

    Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

  11. Geophysical Fluid Flow Cell Simulation

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Computer simulation of atmospheric flow corresponds well to imges taken during the second Geophysical Fluid Flow Cell (BFFC) mission. The top shows a view from the pole, while the bottom shows a view from the equator. Red corresponds to hot fluid rising while blue shows cold fluid falling. This simulation was developed by Anil Deane of the University of Maryland, College Park and Paul Fischer of Argorne National Laboratory. Credit: NASA/Goddard Space Flight Center

  12. A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+ tumor cells

    PubMed Central

    Reusch, Uwe; Duell, Johannes; Ellwanger, Kristina; Herbrecht, Carmen; Knackmuss, Stefan HJ; Fucek, Ivica; Eser, Markus; McAleese, Fionnuala; Molkenthin, Vera; Le Gall, Fabrice; Topp, Max; Little, Melvyn; Zhukovsky, Eugene A

    2015-01-01

    To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19+ cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19+ malignancies with an advantageous safety risk profile and anticipated dosing regimen. PMID:25875246

  13. Interaction force measurement between E. coli cells and nanoparticles immobilized surfaces by using AFM.

    PubMed

    Zhang, Wen; Stack, Andrew G; Chen, Yongsheng

    2011-02-01

    To better understand environmental behaviors of nanoparticles (NPs), we used the atomic force microscopy (AFM) to measure interaction forces between E. coli cells and NPs immobilized on surfaces in an aqueous environment. The results showed that adhesion force strength was significantly influenced by particle size for both hematite (α-Fe(2)O(3)) and corundum (α-Al(2)O(3)) NPs whereas the effect on the repulsive force was not observed. The adhesion force decreased from 6.3±0.7nN to 0.8±0.4nN as hematite NPs increased from 26nm to 98nm in diameter. Corundum NPs exhibited a similar dependence of adhesion force on particle size. The Johnson-Kendall-Roberts (JKR) model was employed to estimate the contact area between E. coli cells and NPs, and based on the JKR model a new model that considers local effective contact area was developed. The prediction of the new model matched the size dependence of adhesion force in experimental results. Size effects on adhesion forces may originate from the difference in local effective contact areas as supported by our model. These findings provide fundamental information for interpreting the environmental behaviors and biological interactions of NPs, which barely have been addressed. PMID:20932723

  14. Interaction force measurement between E. coli cells and nanoparticles immobilized surfaces by using AFM

    SciTech Connect

    Zhang, Wen; Chen, Yongsheng

    2011-01-01

    To better understand environmental behaviors of nanoparticles (NPs), we used the atomic force microscopy (AFM) to measure interaction forces between E. coli cells and NPs immobilized on surfaces in an aqueous environment. The results showed that adhesion force strength was significantly influenced by particle size for both hematite ( -Fe2 O3 ) and corundum ( -Al2 O3 ) NPs whereas the effect on the repulsive force was not observed. The adhesion force decreased from 6.3 0.7 nN to 0.8 0.4 nN as hematite NPs increased from 26 nm to 98 nm in diameter. Corundum NPs exhibited a similar dependence of adhesion force on particle size. The Johnson Kendall Roberts (JKR) model was employed to estimate the contact area between E. coli cells and NPs, and based on the JKR model a new model that considers local effective contact area was developed. The prediction of the new model matched the size dependence of adhesion force in experimental results. Size effects on adhesion forces may originate from the difference in local effective contact areas as supported by our model. These findings provide fundamental information for interpreting the environmental behaviors and biological interactions of NPs, which barely have been addressed.

  15. Probing ternary solvent effect in high Voc polymer solar cells using advanced AFM techniques

    DOE PAGESBeta

    Li, Chao; Soleman, Mikhael; Lorenzo, Josie; Dhasmana, Nitesh; Chantharasupawong, Panit; Ievlev, Anton; Gesquiere, Andre; Tetard, Laurene; Thomas, Jayan

    2016-01-25

    This work describes a simple method to develop a high Voc low band gap PSCs. In addition, two new atomic force microscopy (AFM)-based nanoscale characterization techniques to study the surface morphology and physical properties of the structured active layer are introduced. With the help of ternary solvent processing of the active layer and C60 buffer layer, a bulk heterojunction PSC with Voc more than 0.9 V and conversion efficiency 7.5% is developed. In order to understand the fundamental properties of the materials ruling the performance of the PSCs tested, AFM-based nanoscale characterization techniques including Pulsed-Force-Mode AFM (PFM-AFM) and Mode-Synthesizing AFMmore » (MSAFM) are introduced. Interestingly, MSAFM exhibits high sensitivity for direct visualization of the donor–acceptor phases in the active layer of the PSCs. Lastly, conductive-AFM (cAFM) studies reveal local variations in conductivity in the donor and acceptor phases as well as a significant increase in photocurrent in the PTB7:ICBA sample obtained with the ternary solvent processing.« less

  16. The Emergence of AFM Applications to Cell Biology: How new technologies are facilitating investigation of human cells in health and disease at the nanoscale

    PubMed Central

    Yang, Ruiguo; Xi, Ning; Fung, Carmen Kar Man; Seiffert-Sinha, Kristina; Lai, King Wai Chiu; Sinha, Animesh A.

    2013-01-01

    Atomic Force Microscopy (AFM) based nanorobotics has been used for building nano devices in semiconductors for almost a decade. Leveraging the unparallel precision localization capabilities of this technology, high resolution imaging and mechanical property characterization is now increasingly being performed in biological settings. AFM also offers the prospect for handling and manipulating biological materials at nanometer scale. It has unique advantages over other methods, permitting experiments in the liquid phase where physiological conditions can be maintained. Taking advantage of these properties, our group has visualized membrane and cytoskeletal structures of live cells by controlling the interaction force of the AFM tip with cellular components at the nN or sub-nN range. Cell stiffness changes were observed by statistically analyzing the Young’s modulus values of human keratinocytes before and after specific antibody treatment. Furthermore, we used the AFM cantilever as a robotic arm for mechanical pushing, pulling and cutting to perform nanoscale manipulations of cell-associated structures. AFM guided nano-dissection, or nanosurgery was enacted on the cell in order to sever intermediate filaments connecting neighboring keratinocytes via sub 100 nm resolution cuts. Finally, we have used a functionalized AFM tip to probe cell surface receptors to obtain binding force measurements. This technique formed the basis for Single Molecule Force Spectroscopy (SMFS). In addition to enhancing our basic understanding of dynamic signaling events in cell biology, these advancements in AFM based biomedical investigations can be expected to facilitate the search for biomarkers related to disease diagnosis progress and treatment. PMID:24416719

  17. Adhesion of B. subtilis spores and vegetative cells onto stainless steel--DLVO theories and AFM spectroscopy.

    PubMed

    Harimawan, Ardiyan; Zhong, Shaoping; Lim, Chwee-Teck; Ting, Yen-Peng

    2013-09-01

    Interactions between the bacterium Bacillus subtilis (either as vegetative cells or as spores) and stainless steel 316 (SS-316) surfaces were quantified using the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and extended DLVO (xDLVO) approach in conjunction with live force spectroscopy using an Atomic Force Microscope (AFM). The xDLVO approach accounts for acid-base (polar) interactions that are not considered in the classical DLVO theory. AFM results revealed that spores manifested stronger attraction interactions to stainless steel compared to their vegetative cells counterparts due to lower energy barrier as predicted by both the theoretical approaches as well as the higher hydrophobicity on the spore surfaces. Both DLVO and xDLVO theories predict that vegetative cells manifest weaker attachment on the surfaces compared to spores. Results of AFM force measurement corroborate these findings; spores recorded significantly higher adhesion force (2.92±0.4 nN) compared to vegetative cells (0.65±0.2 nN). The adhesion of spores presents greater challenges in biofilm control owing to its stronger attachment and persistence when the spores are formed under adverse environmental conditions. PMID:23777862

  18. Bioactive compounds immobilized on Ti and TiNbHf: AFM-based investigations of biofunctionalization efficiency and cell adhesion.

    PubMed

    Herranz-Diez, C; Li, Q; Lamprecht, C; Mas-Moruno, C; Neubauer, S; Kessler, H; Manero, J M; Guillem-Martí, J; Selhuber-Unkel, C

    2015-12-01

    Implant materials require optimal biointegration, including strong and stable cell-material interactions from the early stages of implantation. Ti-based alloys with low elastic modulus are attracting a lot of interest for avoiding stress shielding, but their osseointegration potential is still very low. In this study, we report on how cell adhesion is influenced by linear RGD, cyclic RGD, and recombinant fibronectin fragment III8-10 coated on titanium versus a novel low-modulus TiNbHf alloy. The bioactive molecules were either physisorbed or covalently coupled to the substrates and their conformation on the surfaces was investigated with atomic force microscopy (AFM). The influence of the different bioactive coatings on the adhesion of rat mesenchymal stem cells was evaluated using cell culture assays and quantitatively analyzed at the single cell level by AFM-based single-cell force spectroscopy. Our results show that bioactive moieties, particularly fibronectin fragment III8-10, improve cell adhesion on titanium and TiNbHf and that the covalent tethering of such molecules provides the most promising strategy to biofunctionalize these materials. Therefore, the use of recombinant protein fragments is of high importance for improving the osseointegration potential of implant materials. PMID:26513753

  19. AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

    PubMed Central

    Peaucelle, Alexis

    2014-01-01

    We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK AFM. Specifically, in this protocol we measure the apparent Young’s modulus of cell walls at subcellular resolutions across regions of up to 100 µm x 100 µm in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure. In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events. However, several limitations remain: the method can only be used on fairly small samples (around 100 µm in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue’s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future. PMID:25080133

  20. Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

    PubMed

    Acerbi, Irene; Luque, Tomás; Giménez, Alícia; Puig, Marta; Reguart, Noemi; Farré, Ramon; Navajas, Daniel; Alcaraz, Jordi

    2012-01-01

    Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis. PMID:22384196

  1. Local mechanical properties of bladder cancer cells measured by AFM as a signature of metastatic potential

    NASA Astrophysics Data System (ADS)

    Abidine, Y.; Laurent, V. M.; Michel, R.; Duperray, A.; Verdier, C.

    2015-10-01

    The rheological properties of bladder cancer cells of different invasivities have been investigated using a microrheological technique well adapted in the range [1-300Hz] of interest to understand local changes in the cytoskeleton microstructure, in particular actin fibres. Drugs disrupting actin and acto-myosin functions were used to study the resistance of such cancer cells. Results on a variety of cell lines were fitted with a model revealing the importance of two parameters, the elastic shear plateau modulus G N 0 as well as the glassy transition frequency f T. These parameters are good markers for invasiveness, with the notable exception of the cell periphery, which is stiffer for less invasive cells, and could be of importance in cancer metastasis.

  2. Application of the Johnson-Kendall-Roberts model in AFM-based mechanical measurements on cells and gel.

    PubMed

    Efremov, Yu M; Bagrov, D V; Kirpichnikov, M P; Shaitan, K V

    2015-10-01

    The force-distance curves (FCs) obtained by the atomic force microscope (AFM) with colloid probes contain information about both the viscoelastic properties and adhesion of a sample. Here, we processed both the approach and retraction parts of FCs obtained on polyacrylamide gels (in water or PBS) and Vero cells (in a culture medium). The Johnson-Kendall-Roberts model was applied to the retraction curves to account for the adhesion. The effects of loading rate, holding time and indentation depth on adhesion force and Young's modulus, calculated from approach and retraction curves, were studied. It was shown that both bulk and local interfacial viscoelasticity can affect the observed approach-retraction hysteresis and measured parameters. The addition of 1% bovine serum albumin (BSA) decreased adhesion of the probe to the PAA gel surface, so interfacial viscoelasticity effects were diminished. On the contrary, the adhesiveness of Vero cells increased after BSA addition, indicating the complex nature of the cell-probe interaction. PMID:26186106

  3. Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves.

    PubMed

    Digiuni, Simona; Berne-Dedieu, Annik; Martinez-Torres, Cristina; Szecsi, Judit; Bendahmane, Mohammed; Arneodo, Alain; Argoul, Françoise

    2015-05-01

    Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions. PMID:25954881

  4. Single Cell Wall Nonlinear Mechanics Revealed by a Multiscale Analysis of AFM Force-Indentation Curves

    PubMed Central

    Digiuni, Simona; Berne-Dedieu, Annik; Martinez-Torres, Cristina; Szecsi, Judit; Bendahmane, Mohammed; Arneodo, Alain; Argoul, Françoise

    2015-01-01

    Individual plant cells are rather complex mechanical objects. Despite the fact that their wall mechanical strength may be weakened by comparison with their original tissue template, they nevertheless retain some generic properties of the mother tissue, namely the viscoelasticity and the shape of their walls, which are driven by their internal hydrostatic turgor pressure. This viscoelastic behavior, which affects the power-law response of these cells when indented by an atomic force cantilever with a pyramidal tip, is also very sensitive to the culture media. To our knowledge, we develop here an original analyzing method, based on a multiscale decomposition of force-indentation curves, that reveals and quantifies for the first time the nonlinearity of the mechanical response of living single plant cells upon mechanical deformation. Further comparing the nonlinear strain responses of these isolated cells in three different media, we reveal an alteration of their linear bending elastic regime in both hyper- and hypotonic conditions. PMID:25954881

  5. Combined single cell AFM manipulation and TIRFM for probing the molecular stability of multilayer fibrinogen matrices

    PubMed Central

    Christenson, W.; Yermolenko, I.; Plochberger, B.; Camacho-Alanis, F.; Ros, A.; Ugarova, T.P.; Ros, R.

    2014-01-01

    Adsorption of fibrinogen on various surfaces produces a nanoscale multilayer matrix, which strongly reduces the adhesion of platelets and leukocytes with implications for hemostasis and blood compatibility of biomaterials. The nonadhesive properties of fibrinogen matrices are based on their extensibility, ensuing the inability to transduce strong mechanical forces via cellular integrins and resulting in weak intracellular signaling. In addition, reduced cell adhesion may arise from the weaker associations between fibrinogen molecules in the superficial layers of the matrix. Such reduced stability would allow integrins to pull fibrinogen molecules out of the matrix with comparable or smaller forces than required to break integrin–fibrinogen bonds. To examine this possibility, we developed a method based on the combination of total internal reflection fluorescence microscopy, single cell manipulation with an atomic force microscope and microcontact printing to study the transfer of fibrinogen molecules out of a matrix onto cells. We calculated the average fluorescence intensities per pixel for wild-type HEK 293 (HEK WT) and HEK 293 cells expressing leukocyte integrin Mac-1 (HEK Mac-1) before and after contact with multilayered matrices of fluorescently labeled fibrinogen. For contact times of 500 s, HEK Mac-1 cells show a median increase of 57% of the fluorescence intensity compared to 6% for HEKWT cells. The results suggest that the integrin Mac-1-fibrinogen interactions are stronger than the intermolecular fibrinogen interactions in the superficial layer of the matrix. The low mechanical stability of the multilayer fibrinogen surface may contribute to the reduced cell adhesive properties of fibrinogen-coated substrates. We anticipate that the described method can be applied to various cell types to examine their integrin-mediated adhesion to the extracellular matrices with a variable protein composition. PMID:24239757

  6. Combining AFM and Acoustic Probes to Reveal Changes in the Elastic Stiffness Tensor of Living Cells

    PubMed Central

    Nijenhuis, Nadja; Zhao, Xuegen; Carisey, Alex; Ballestrem, Christoph; Derby, Brian

    2014-01-01

    Knowledge of how the elastic stiffness of a cell affects its communication with its environment is of fundamental importance for the understanding of tissue integrity in health and disease. For stiffness measurements, it has been customary to quote a single parameter quantity, e.g., Young’s modulus, rather than the minimum of two terms of the stiffness tensor required by elasticity theory. In this study, we use two independent methods (acoustic microscopy and atomic force microscopy nanoindentation) to characterize the elastic properties of a cell and thus determine two independent elastic constants. This allows us to explore in detail how the mechanical properties of cells change in response to signaling pathways that are known to regulate the cell’s cytoskeleton. In particular, we demonstrate that altering the tensioning of actin filaments in NIH3T3 cells has a strong influence on the cell's shear modulus but leaves its bulk modulus unchanged. In contrast, altering the polymerization state of actin filaments influences bulk and shear modulus in a similar manner. In addition, we can use the data to directly determine the Poisson ratio of a cell and show that in all cases studied, it is less than, but very close to, 0.5 in value. PMID:25296302

  7. Human epithelial cancer cells studied using combined AFM-IR absorption nanoimaging

    NASA Astrophysics Data System (ADS)

    Kennedy, Eamonn; Al-Majomaie, Rasoul; Zerulla, Dominic; Al-Rubeai, Mohammed; Rice, James H.

    2014-05-01

    Several recent studies have described the use of infrared (IR) nanoimaging for non-invasive chemical discrimination of subcellular features and intracellular exogenous agents. In this work we outline a number of improvements in both quantitative IR nanoimage analysis and optical system improvements which enable recovery of nanoscale subcellular chemical localization with improved chemical precision. Additionally, we demonstrate how a combination of IR absorption nanoimaging and topographic data can produce subcellular chemical density and complexity maps, which can illustrate several cellular features of interest, including the label free localization of nuclei for both healthy and cancerous cell lines with sub 40nm accuracy. As many cell processes related to disease are governed by the position and dynamics of subcellular features, we present the ability to map biochemical inhomogeneity of cancer cells at nanoscale resolution as a means to explore the subcellular biomechanics underlying carcinogenesis.

  8. Fuel cell membrane hydration and fluid metering

    DOEpatents

    Jones, Daniel O.; Walsh, Michael M.

    1999-01-01

    A hydration system includes fuel cell fluid flow plate(s) and injection port(s). Each plate has flow channel(s) with respective inlet(s) for receiving respective portion(s) of a given stream of reactant fluid for a fuel cell. Each injection port injects a portion of liquid water directly into its respective flow channel in order to mix its respective portion of liquid water with the corresponding portion of the stream. This serves to hydrate at least corresponding part(s) of a given membrane of the corresponding fuel cell(s). The hydration system may be augmented by a metering system including flow regulator(s). Each flow regulator meters an injecting at inlet(s) of each plate of respective portions of liquid into respective portion(s) of a given stream of fluid by corresponding injection port(s).

  9. Fuel cell membrane hydration and fluid metering

    DOEpatents

    Jones, Daniel O.; Walsh, Michael M.

    2003-01-01

    A hydration system includes fuel cell fluid flow plate(s) and injection port(s). Each plate has flow channel(s) with respective inlet(s) for receiving respective portion(s) of a given stream of reactant fluid for a fuel cell. Each injection port injects a portion of liquid water directly into its respective flow channel. This serves to hydrate at least corresponding part(s) of a given membrane of the corresponding fuel cell(s). The hydration system may be augmented by a metering system including flow regulator(s). Each flow regulator meters an injecting at inlet(s) of each plate of respective portions of liquid into respective portion(s) of a given stream of fluid by corresponding injection port(s).

  10. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells.

    PubMed

    Gavara, Núria

    2016-01-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies. PMID:26891762

  11. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

    PubMed Central

    Gavara, Núria

    2016-01-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies. PMID:26891762

  12. WGA-QD probe-based AFM detects WGA-binding sites on cell surface and WGA-induced rigidity alternation

    SciTech Connect

    Wang Xiaoping; He Dongmei; Cai Jiye Chen Tongsheng; Zou Feiyan; Li Yalan; Wu Yangzhe; Chen, Zheng W.; Chen Yong

    2009-02-06

    A strategy involving the conjugation of fluorescent quantum dot (QD) with wheat germ agglutinin (WGA) acting as fluorescent and topographic probes prior to cell surface staining is developed for fluorescence microscopy and atomic force microscopy (AFM). This strategy provided at least two advantages: (a) an amplified fluorescence of WGA-QD aggregates, strongly resistant to photobleaching, ensures repeated/real-time observations of the probe-labeled cells by fluorescence microscopy; (b) the enlarged size of WGA-QD probe makes it possible for labeled WGA to be distinguished from other membrane proteins by AFM. Here, the random distribution of WGA-binding sites on non-crosslinked cells and the uneven or polarized reorganization due to WGA-induced crosslinking on cell surfaces were studied using AFM-detectable WGA-QD probe. Moreover, we developed a method to rapidly detect the WGA-induced rigidity alternation of the whole cells, which is efficient and has the potentiality of being developed to a useful tool in clinical diagnosis.

  13. Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

    NASA Astrophysics Data System (ADS)

    Gavara, Núria

    2016-02-01

    Atomic Force Microscopy (AFM) is a widely used tool to study cell mechanics. Current AFM setups perform high-throughput probing of living cells, generating large amounts of force-indentations curves that are subsequently analysed using a contact-mechanics model. Here we present several algorithms to detect the contact point in force-indentation curves, a crucial step to achieve fully-automated analysis of AFM-generated data. We quantify and rank the performance of our algorithms by analysing a thousand force-indentation curves obtained on thin soft homogeneous hydrogels, which mimic the stiffness and topographical profile of adherent cells. We take advantage of the fact that all the proposed algorithms are based on sequential search strategies, and show that a combination of them yields the most accurate and unbiased results. Finally, we also observe improved performance when force-indentation curves obtained on adherent cells are analysed using our combined strategy, as compared to the classical algorithm used in the majority of previous cell mechanics studies.

  14. Fluid-fluid levels in giant cell tumors of bone: report of two cases.

    PubMed

    Kaplan, P A; Murphey, M; Greenway, G; Resnick, D; Sartoris, D J; Harms, S

    1987-04-01

    Fluid-fluid levels have been described in association with aneurysmal bone cysts, telangiectatic osteosarcoma, and a chondroblastoma. We report two cases of giant cell tumors of bone with fluid-fluid levels identified by computed tomography and, in one case, by magnetic resonance imaging. This finding has not previously been associated with giant cell tumors. The radiographic features of the fluid-fluid levels cannot be distinguished from those reported in other osseous neoplasms. PMID:3581850

  15. AFM analysis of the multiple types of molecular interactions involved in rituximab lymphoma therapy on patient tumor cells and NK cells.

    PubMed

    Li, Mi; Xiao, Xiubin; Zhang, Weijing; Liu, Lianqing; Xi, Ning; Wang, Yuechao

    2014-08-01

    Rituximab is a monoclonal antibody drug approved for the treatment of patients with lymphomas. Rituximab's main killing mechanism is antibody-dependent cellular cytotoxicity (ADCC). During ADCC, rituximab's fragment antigen binding (Fab) region binds to the CD20 antigen on the tumor cell and its fragment crystallizable (Fc) region binds to the Fc receptor (FcR) on the natural killer (NK) cells. In this study, two types of molecular interactions (CD20-rituximab, FcR-rituximab) involved in ADCC were measured simultaneously on cells prepared from biopsy specimens of lymphoma patients by utilizing atomic force microscopy (AFM) with functionalized tips carrying rituximab. NK cells were detected by specific NKp46 fluorescent labeling and tumor cells were detected by specific ROR1 fluorescent labeling. Based on the fluorescence recognition, the binding affinity and distribution of FcRs on NK cells, and CD20 on tumor cells, were quantitatively measured and mapped. The binding affinity and distribution of FcRs (on NK cells) and CD20 (on tumor cells) were associated with rituximab clinical efficacy. The experimental results provide a new approach to simultaneously quantify the multiple types of molecular interactions involved in rituximab ADCC mechanism on patient biopsy cells, which is of potential clinical significance to predict rituximab efficacy for personalized medicine. PMID:25117605

  16. The Geophysical Fluid Flow Cell Experiment

    NASA Technical Reports Server (NTRS)

    Hart, J. E.; Ohlsen, D.; Kittleman, S.; Borhani, N.; Leslie, F.; Miller, T.

    1999-01-01

    The Geophysical Fluid Flow Cell (GFFC) experiment performed visualizations of thermal convection in a rotating differentially heated spherical shell of fluid. In these experiments dielectric polarization forces are used to generate a radially directed buoyancy force. This enables the laboratory simulation of a number of geophysically and astrophysically important situations in which sphericity and rotation both impose strong constraints on global scale fluid motions. During USML-2 a large set of experiments with spherically symmetric heating were carried out. These enabled the determination of critical points for the transition to various forms of nonaxisymmetric convection and, for highly turbulent flows, the transition latitudes separating the different modes of motion. This paper presents a first analysis of these experiments as well as data on the general performance of the instrument during the USML-2 flight.

  17. Successive detection of insulin-like growth factor-II bound to receptors on a living cell surface using an AFM.

    PubMed

    Han, Sung-Woong; Mieda, Shingo; Nakamura, Chikashi; Kihara, Takanori; Nakamura, Noriyuki; Miyake, Jun

    2011-01-01

    In this study, we have developed a method of mechanical force detection for ligands bound to receptors on a cell surface, both of which are involved in a signal transduction pathway. This pathway is an autocrine pathway, involving the production of insulin-like growth factor-II (IGF-II) and activation of the IGF-I receptor, involved in myoblast differentiation induced by MyoD in C3H10T1/2 mouse mesenchymal stem cells. Differentiation of C3H10T1/2 was induced with the DNA demethylation agent 5-azacytidine (5-aza). The etched AFM tip used in the force detection had a flat surface of which about 10 µm(2) was in contact with a cell surface. The forces required to rupture the interactions of IGF-IIs on a cell and anti mouse IGF-II polyclonal antibody immobilized on an etched AFM tip were measured within 5 days of induction of differentiation. The mean unbinding force for a single paired antibody-ligand on a cell was about 81 pN, which was measured at a force loading rate of about 440 nN/s. The percentage of unbinding forces over 100 pN increased to 32% after 2 days from the addition of 5-aza to the medium. This method could be used in non-invasive and successive evaluation of a living cell's behavior. PMID:19953597

  18. Geophysical Fluid Flow Cell (GFFC) Simulation

    NASA Technical Reports Server (NTRS)

    1999-01-01

    These simulations of atmospheric flow use the same experimental parameters but started with slightly different initial conditions in the model. The simulations were part of data analysis for the Geophysical Fluid Flow Cell (GFFC), a planet in a test tube apparatus flown on Spacelab to mimic the atmospheres on gas giant planets and stars. (Credit: Dr. Tim Miller of Global Hydrology and Climate Center at the Marshall Space Flight Center)

  19. A Multifunctional Frontloading Approach for Repeated Recycling of a Pressure-Controlled AFM Micropipette.

    PubMed

    Roder, Phillip; Hille, Carsten

    2015-01-01

    Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few μL using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy. PMID:26636981

  20. A Multifunctional Frontloading Approach for Repeated Recycling of a Pressure-Controlled AFM Micropipette

    PubMed Central

    Roder, Phillip; Hille, Carsten

    2015-01-01

    Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few μL using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy. PMID:26636981

  1. Qplus AFM driven nanostencil.

    PubMed

    Grévin, B; Fakir, M; Hayton, J; Brun, M; Demadrille, R; Faure-Vincent, J

    2011-06-01

    We describe the development of a novel setup, in which large stencils with suspended silicon nitride membranes are combined with atomic force microscopy (AFM) regulation by using tuning forks. This system offers the possibility to perform separate AFM and nanostencil operations, as well as combined modes when using stencil chips with integrated tips. The flexibility and performances are demonstrated through a series of examples, including wide AFM scans in closed loop mode, probe positioning repeatability of a few tens of nanometer, simultaneous evaporation of large (several hundred of micron square) and nanoscopic metals and fullerene patterns in static, multistep, and dynamic modes. This approach paves the way for further developments, as it fully combines the advantages of conventional stenciling with the ones of an AFM driven shadow mask. PMID:21721701

  2. Two-Dimensional Measurement of n+-p Asymmetrical Junctions in Multicrystalline Silicon Solar Cells Using AFM-Based Electrical Techniques with Nanometer Resolution: Preprint

    SciTech Connect

    Jiang, C. S.; Moutinho, H. R.; Li, J. V.; Al-Jassim, M. M.; Heath, J. T.

    2011-07-01

    Lateral inhomogeneities of modern solar cells demand direct electrical imaging with nanometer resolution. We show that atomic force microscopy (AFM)-based electrical techniques provide unique junction characterizations, giving a two-dimensional determination of junction locations. Two AFM-based techniques, scanning capacitance microscopy/spectroscopy (SCM/SCS) and scanning Kelvin probe force microscopy (SKPFM), were significantly improved and applied to the junction characterizations of multicrystalline silicon (mc-Si) cells. The SCS spectra were taken pixel by pixel by precisely controlling the tip positions in the junction area. The spectra reveal distinctive features that depend closely on the position relative to the electrical junction, which allows us to indentify the electrical junction location. In addition, SKPFM directly probes the built-in potential over the junction area modified by the surface band bending, which allows us to deduce the metallurgical junction location by identifying a peak of the electric field. Our results demonstrate resolutions of 10-40 nm, depending on the techniques (SCS or SKPFM). These direct electrical measurements with nanometer resolution and intrinsic two-dimensional capability are well suited for investigating the junction distribution of solar cells with lateral inhomogeneities.

  3. Supercritical Fluid Facilitated Disintegration of Hexagonal Boron Nitride Nanosheets to Quantum Dots and Its Application in Cells Imaging.

    PubMed

    Thangasamy, Pitchai; Santhanam, Manikandan; Sathish, Marappan

    2016-07-27

    Preparation of quantum dots (QDs) and exfoliation of two-dimensional layered materials have gathered significant attention in recent days. Though, there are number of attempts have been reported, facile and efficient methodology is yet to be explored. Here, we demonstrate supercritical fluid processing approach for rapid and facile synthesis of blue luminescent BN QDs from layered bulk material via in situ exfoliation followed by disintegration. The microscopic and AFM analysis confirmed the few layer BN QDs formation. The strong luminescent behavior of BN QDs is utilized to stain Gram-negative bacterial cells specifically in the presence of Gram-positive bacterial cells. PMID:27391298

  4. Geophysical Fluid Flow Cell (GFFC) Cross Section

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This drawing shows a cross-section view of the test cell at the heart of the Geophysical Fluid Flow Cell (GFFC) that flew on two Spacelab missions. The middle and lower drawings depict the volume of the silicone oil layer that served as the atmosphere as the steel ball rotated and an electrostatic field pulled the oil inward to mimic gravity's effects during the experiments. The GFFC thus produced flow patterns that simulated conditions inside the atmospheres of Jupiter and the Sun and other stars. The principal investigator was John Hart of the University of Colorado at Boulder. It was managed by NASA's Marshall Space Flight Center (MSFC). An Acrobat PDF copy of this drawing is available at http://microgravity.nasa.gov/gallery. (Credit: NASA/Marshall Space Flight Center)

  5. Amniotic fluid stem cells prevent β-cell injury

    PubMed Central

    VILLANI, VALENTINA; MILANESI, ANNA; SEDRAKYAN, SARGIS; DA SACCO, STEFANO; ANGELOW, SUSANNE; CONCONI, MARIA TERESA; DI LIDDO, ROSA; DE FILIPPO, ROGER; PERIN, LAURA

    2015-01-01

    Background aims The contribution of amniotic fluid stem cells (AFSC) to tissue protection and regeneration in models of acute and chronic kidney injuries and lung failure has been shown in recent years. In the present study, we used a chemically induced mouse model of type 1 diabetes to determine whether AFSC could play a role in modulating β-cell injury and restoring β-cell function. Methods Streptozotocin-induced diabetic mice were given intracardial injection of AFSC; morphological and physiological parameters and gene expression profile for the insulin pathway were evaluated after cell transplantation. Results AFSC injection resulted in protection from β-cell damage and increased β-cell regeneration in a subset of mice as indicated by glucose and insulin levels, increased islet mass and preservation of islet structure. Moreover, β-cell preservation/regeneration correlated with activation of the insulin receptor/Pi3K/Akt signaling pathway and vascular endothelial growth factor-A expression involved in maintaining β-cell mass and function. Conclusions Our results suggest a therapeutic role for AFSC in preserving and promoting endogenous β-cell functionality and proliferation. The protective role of AFSC is evident when stem cell transplantation is performed before severe hyperglycemia occurs, which suggests the importance of early intervention. The present study demonstrates the possible benefits of the application of a non–genetically engineered stem cell population derived from amniotic fluid for the treatment of type 1 diabetes mellitus and gives new insight on the mechanism by which the beneficial effect is achieved. PMID:24210784

  6. Fluid flow plate for decreased density of fuel cell assembly

    DOEpatents

    Vitale, Nicholas G.

    1999-01-01

    A fluid flow plate includes first and second outward faces. Each of the outward faces has a flow channel thereon for carrying respective fluid. At least one of the fluids serves as reactant fluid for a fuel cell of a fuel cell assembly. One or more pockets are formed between the first and second outward faces for decreasing density of the fluid flow plate. A given flow channel can include one or more end sections and an intermediate section. An interposed member can be positioned between the outward faces at an interface between an intermediate section, of one of the outward faces, and an end section, of that outward face. The interposed member can serve to isolate the reactant fluid from the opposing outward face. The intermediate section(s) of flow channel(s) on an outward face are preferably formed as a folded expanse.

  7. [AFM fishing of proteins under impulse electric field].

    PubMed

    Ivanov, Yu D; Pleshakova, T O; Malsagova, K A; Kaysheva, A L; Kopylov, A T; Izotov, A A; Tatur, V Yu; Vesnin, S G; Ivanova, N D; Ziborov, V S; Archakov, A I

    2016-05-01

    A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell. PMID:27562998

  8. Stem cells from amniotic fluid - Potential for regenerative medicine.

    PubMed

    Loukogeorgakis, Stavros P; De Coppi, Paolo

    2016-02-01

    Regenerative medicine has recently been established as an emerging field focussing on repair, replacement or regeneration of cells, tissues and whole organs. The significant recent advances in the field have intensified the search for novel sources of stem cells with potential for therapy. Recently, researchers have identified the amniotic fluid as an untapped source of stem cells that are multipotent, possess immunomodulatory properties and do not have the ethical and legal limitations of embryonic stem cells. Stem cells from the amniotic fluid have been shown to differentiate into cell lineages representing all three embryonic germ layers without generating tumours, which make them an ideal candidate for tissue engineering applications. In addition, their ability to engraft in injured organs and modulate immune and repair responses of host tissues suggest that transplantation of such cells may be useful for the treatment of various degenerative and inflammatory diseases affecting major tissues/organs. This review summarises the evidence on amniotic fluid cells over the past 15 years and explores the potential therapeutic applications of amniotic fluid stem cells and amniotic fluid mesenchymal stem cells. PMID:26542929

  9. Contact nanomechanical measurements with the AFM

    NASA Astrophysics Data System (ADS)

    Geisse, Nicholas

    2013-03-01

    The atomic force microscope (AFM) has found broad use in the biological sciences largely due to its ability to make measurements on unfixed and unstained samples under liquid. In addition to imaging at multiple spatial scales ranging from micro- to nanometer, AFMs are commonly used as nanomechanical probes. This is pertinent for cell biology, as it has been demonstrated that the geometrical and mechanical properties of the extracellular microenvironment are important in such processes as cancer, cardiovascular disease, muscular dystrophy, and even the control of cell life and death. Indeed, the ability to control and quantify these external geometrical and mechanical parameters arises as a key issue in the field. Because AFM can quantitatively measure the mechanical properties of various biological samples, novel insights to cell function and to cell-substrate interactions are now possible. As the application of AFM to these types of problems is widened, it is important to understand the performance envelope of the technique and its associated data analyses. This talk will discuss the important issues that must be considered when mechanical models are applied to real-world data. Examples of the effect of different model assumptions on our understanding of the measured material properties will be shown. Furthermore, specific examples of the importance of mechanical stimuli and the micromechanical environment to the structure and function of biological materials will be presented.

  10. Atomic Force Microscopy of Living Cells

    NASA Astrophysics Data System (ADS)

    Ushiki, Tatsuo; Yamamoto, Susumu; Hitomi, Jiro; Ogura, Shigeaki; Umemoto, Takeshi; Shigeno, Masatsugu

    2000-06-01

    This paper is a review of our results of the application of atomic force microscopy (AFM) to the three-dimensional observation of living cells. First, we showed AFM images of living cultured cells in fluid. Contact mode AFM of living cells provided precise information on the shape of cellular processes (such as spike-like processes or lamellipodia) at the cellular margin. The contour of cytoskeletal elements just beneath the cell membrane was also clearly observable on the upper surface of the cells. Secondly, we showed the data on the discrepancy between the AFM images of living cells and fixed cells. These findings were useful for evaluating AFM images of living cells. Finally, we described the time-lapse AFM of living cells. A fluid chamber system enabled us to obtain AFM images of living cells for over 1 h at time intervals of 2-4 min. A series of these AFM images were useful for examining the movements of cellular processes in relation to subcellular cytoskeletal elements. Time-lapse movies produced by sequential AFM images also gave a realistic view of the cellular dynamics.

  11. Effects of Fluid Shear Stress on Cancer Stem Cell Viability

    NASA Astrophysics Data System (ADS)

    Sunday, Brittney; Triantafillu, Ursula; Domier, Ria; Kim, Yonghyun

    2014-11-01

    Cancer stem cells (CSCs), which are believed to be the source of tumor formation, are exposed to fluid shear stress as a result of blood flow within the blood vessels. It was theorized that CSCs would be less susceptible to cell death than non-CSCs after both types of cell were exposed to a fluid shear stress, and that higher levels of fluid shear stress would result in lower levels of cell viability for both cell types. To test this hypothesis, U87 glioblastoma cells were cultured adherently (containing smaller populations of CSCs) and spherically (containing larger populations of CSCs). They were exposed to fluid shear stress in a simulated blood flow through a 125-micrometer diameter polyetheretherketone (PEEK) tubing using a syringe pump. After exposure, cell viability data was collected using a BioRad TC20 Automated Cell Counter. Each cell type was tested at three physiological shear stress values: 5, 20, and 60 dynes per centimeter squared. In general, it was found that the CSC-enriched U87 sphere cells had higher cell viability than the CSC-depleted U87 adherent cancer cells. Interestingly, it was also observed that the cell viability was not negatively affected by the higher fluid shear stress values in the tested range. In future follow-up studies, higher shear stresses will be tested. Furthermore, CSCs from different tumor origins (e.g. breast tumor, prostate tumor) will be tested to determine cell-specific shear sensitivity. National Science Foundation Grant #1358991 supported the first author as an REU student.

  12. Fluid shear stress modulation of hepatocyte-like cell function.

    PubMed

    Rashidi, Hassan; Alhaque, Sharmin; Szkolnicka, Dagmara; Flint, Oliver; Hay, David C

    2016-07-01

    Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype. PMID:26979076

  13. Fluid shear, intercellular stress, and endothelial cell alignment

    PubMed Central

    Steward, Robert; Tambe, Dhananjay; Hardin, C. Corey; Krishnan, Ramaswamy

    2015-01-01

    Endothelial cell alignment along the direction of laminar fluid flow is widely understood to be a defining morphological feature of vascular homeostasis. While the role of associated signaling and structural events have been well studied, associated intercellular stresses under laminar fluid shear have remained ill-defined and the role of these stresses in the alignment process has remained obscure. To fill this gap, we report here the tractions as well as the complete in-plane intercellular stress fields measured within the human umbilical vein endothelial cell (HUVEC) monolayer subjected to a steady laminar fluid shear of 1 Pa. Tractions, intercellular stresses, as well as their time course, heterogeneity, and anisotropy, were measured using monolayer traction microscopy and monolayer stress microscopy. Prior to application of laminar fluid flow, intercellular stresses were largely tensile but fluctuated dramatically in space and in time (317 ± 122 Pa). Within 12 h of the onset of laminar fluid flow, the intercellular stresses decreased substantially but continued to fluctuate dramatically (142 ± 84 Pa). Moreover, tractions and intercellular stresses aligned strongly and promptly (within 1 h) along the direction of fluid flow, whereas the endothelial cell body aligned less strongly and substantially more slowly (12 h). Taken together, these results reveal that steady laminar fluid flow induces prompt reduction in magnitude and alignment of tractions and intercellular stress tensor components followed by the retarded elongation and alignment of the endothelial cell body. Appreciably smaller intercellular stresses supported by cell-cell junctions logically favor smaller incidence of gap formation and thus improved barrier integrity. PMID:25652451

  14. Alternative experiments using the geophysical fluid flow cell

    NASA Technical Reports Server (NTRS)

    Hart, J. E.

    1984-01-01

    This study addresses the possibility of doing large scale dynamics experiments using the Geophysical Fluid Flow Cell. In particular, cases where the forcing generates a statically stable stratification almost everywhere in the spherical shell are evaluated. This situation is typical of the Earth's atmosphere and oceans. By calculating the strongest meridional circulation expected in the spacelab experiments, and testing its stability using quasi-geostrophic stability theory, it is shown that strongly nonlinear baroclinic waves on a zonally symmetric modified thermal wind will not occur. The Geophysical Fluid Flow Cell does not have a deep enough fluid layer to permit useful studies of large scale planetary wave processes arising from instability. It is argued, however, that by introducing suitable meridional barriers, a significant contribution to the understanding of the oceanic thermocline problem could be made.

  15. Selective activation of functional suppressor cells by human seminal fluid.

    PubMed Central

    Witkin, S S

    1986-01-01

    The ability of seminal fluid (SF) to induce suppressor cell activity from peripheral blood mononuclear cells (PBMN) was examined. PBMN were incubated with SF for 48 h, washed to remove SF components, treated with mitomycin C (mit C) and co-cultured with Raji cells, a lymphoblastoid cell line. Raji cell proliferation was inhibited by SF-treated PBMN proportionally to SF concentration. SF (50-200 micrograms), mit C-treated Raji cells or mit C-treated PBMN pre-incubated with phytohaemagglutinin were without effect on Raji cell growth. Suppressor T lymphocytes generated by incubation of PBMN with concanavalin A inhibited Raji cells to the same extent as did SF-treated PBMN. All activity was lost following heating at 56 degrees C for 30 min; freezing and thawing reduced the ability of SF to induce suppression by 50%. Dialysis of SF or treatment with antibody to prostaglandin E2 led to a 50% reduction in suppression. PMID:2943541

  16. Chondrogenic differentiation of amniotic fluid-derived stem cells.

    PubMed

    Kolambkar, Yash M; Peister, Alexandra; Soker, Shay; Atala, Anthony; Guldberg, Robert E

    2007-10-01

    For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible, can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-beta (TGF-beta) supplementation, with TGF-beta1 producing greater increases than TGF-beta3. Modification of expansion media supplements and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-beta1 supplementation alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three weeks of TGF-beta1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications. PMID:17668282

  17. Fluid and Cell Transport Through a Microfabricated Flow Chamber.

    NASA Astrophysics Data System (ADS)

    Brody, James Patrick

    We use silicon processing techniques to construct microfabricated fluid flow chambers. Custom designed silicon wafers with feature sizes of 1-10 μm and etch depths from 0.5-5 μm are anodically bonded to Pyrex glass to create a hermetically sealed chamber. A pressure gradient is placed across the chamber to induce bulk fluid flow. Properties of fluid flow and red blood cells are recorded using video microscopy. The human red blood cell is ideal for studying cellular membranes. It is an 8 μm diameter biconcave disc containing a membrane and associated cytoskeleton which surrounds a thick solution of hemoglobin. The material properties of individual red blood cells have been extensively studied in the past using micropipettes. However, we can get statistics on hundreds of red blood cells by fabricating an array of narrow channels 4 mu m x 4 μm in cross-section (the diameter of the smallest capillaries in the human body) and 13 μm long. These narrow channels are followed by an open space. This geometry forces red cells to repeatedly fold and unfold. Using these arrays, we show that the shear modulus of the membrane does not have a unique value, but has a distribution that ranges from 3-12 times 10 ^{-6} N/m. The surprisingly wide distribution is not due to cell size or cell age. It does seem to be correlated with intracellular Ca^ {2+}<=vels, leading us to believe that cell rigidity is controlled by some active process. We also report observations on red blood cells changing their rigidity by factors of fifty over tens of seconds. These microfabricated flow chambers are ideal for studying fluid flow through porous media. We construct custom designed two-dimensional environments with micron size features. These environments can be described by simple analytical theories which also attempt to describe flow through rock. For example, we image viscous imbibition of water into a percolation grid with 5 mu m edges in real time, and measure the permeability as a function

  18. PREFACE: Non-contact AFM Non-contact AFM

    NASA Astrophysics Data System (ADS)

    Giessibl, Franz J.; Morita, Seizo

    2012-02-01

    This special issue is focussed on high resolution non-contact atomic force microscopy (AFM). Non-contact atomic force microscopy was established approximately 15 years ago as a tool to image conducting and insulating surfaces with atomic resolution. Since 1998, an annual international conference has taken place, and although the proceedings of these conferences are a useful source of information, several key developments warrant devoting a special issue to this subject. In the theoretic field, the possibility of supplementing established techniques such as scanning tunneling microscopy (STM) and Kelvin probe microscopy with atomically resolved force micrsoscopy poses many challenges in the calculation of contrast and contrast reversal. The surface science of insulators, self-assembled monolayers and adsorbates on insulators is a fruitful field for the application of non-contact AFM: several articles in this issue are devoted to these subjects. Atomic imaging and manipulation have been pioneered using STM, but because AFM allows the measurement of forces, AFM has had a profound impact in this field as well. Three-dimensional force spectroscopy has allowed many important insights into surface science. In this issue a combined 3D tunneling and force microscopy is introduced. Non-contact AFM typically uses frequency modulation to measure force gradients and was initially used mainly in a vacuum. As can be seen in this issue, frequency modulation is now also used in ambient conditions, allowing better spatial and force resolution. We thank all of the contributors for their time and efforts in making this special issue possible. We are also very grateful to the staff of IOP Publishing for handling the administrative aspects and for steering the refereeing process. Non-contact AFM contents Relation between the chemical force and the tunnelling current in atomic point contacts: a simple model Pavel Jelínek, Martin Ondrácek and Fernando Flores Theoretical simulation of

  19. Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

    SciTech Connect

    Aoki, Shigehisa; Ikeda, Satoshi; Takezawa, Toshiaki; Kishi, Tomoya; Makino, Junichi; Uchihashi, Kazuyoshi; Matsunobu, Aki; Noguchi, Mitsuru; Sugihara, Hajime; Toda, Shuji

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Late-onset peritoneal fibrosis leading to EPS remains to be elucidated. Black-Right-Pointing-Pointer Fluid streaming is a potent factor for peritoneal fibrosis in PD. Black-Right-Pointing-Pointer We focused on the prolonged effect of fluid streaming on mesothelial cell kinetics. Black-Right-Pointing-Pointer A history of fluid streaming exposure promoted mesothelial proliferative activity. Black-Right-Pointing-Pointer We have thus identified a potent new factor for late-onset peritoneal fibrosis. -- Abstract: Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed

  20. Effect of tip mass on frequency response and sensitivity of AFM cantilever in liquid.

    PubMed

    Farokh Payam, Amir; Fathipour, Morteza

    2015-03-01

    The effect of tip mass on the frequency response and sensitivity of atomic force microscope (AFM) cantilever in the liquid environment is investigated. For this purpose, using Euler-Bernoulli beam theory and considering tip mass and hydrodynamic functions in a liquid environment, an expression for the resonance frequencies of AFM cantilever in liquid is derived. Then, based on this expression, the effect of the surface contact stiffness on the flexural mode of a rectangular AFM cantilever in fluid is investigated and compared with the case where the AFM cantilever operates in the air. The results show that in contrast with an air environment, the tip mass has no significant impact on the resonance frequency and sensitivity of the AFM cantilever in the liquid. Hence, analysis of AFM behaviour in liquid environment by neglecting the tip mass is logical. PMID:25562584

  1. Multilineage potential research of bovine amniotic fluid mesenchymal stem cells.

    PubMed

    Gao, Yuhua; Zhu, Zhiqiang; Zhao, Yuhua; Hua, Jinlian; Ma, Yuehui; Guan, Weijun

    2014-01-01

    The use of amnion and amniotic fluid (AF) are abundant sources of mesenchymal stem cells (MSCs) that can be harvested at low cost and do not pose ethical conflicts. In human and veterinary research, stem cells derived from these tissues are promising candidates for disease treatment, specifically for their plasticity, their reduced immunogenicity, and high anti-inflammatory potential. This work aimed to obtain and characterize bovine amniotic fluid mesenchymal stem cells (AFMSC). The bovine AF from the amniotic cavity of pregnant gilts in the early stages of gestation (3- and 4-m-old bovine embryos) was collected. AFMSCs exhibit a fibroblastic-like morphology only starting from the fourth passage, being heterogeneous during the primary culture. Immunofluorescence results showed that AFMSCs were positive for β-integrin, CD44, CD73 and CD166, but negative for CD34, CD45. Meanwhile, AFMSCs expressed ES cell markers, such as Oct4, and when appropriately induced, are capable of differentiating into ectodermal and mesodermal lineages. This study reinforces the emerging importance of these cells as ideal tools in veterinary medicine; future studies aimed at a deeper evaluation of their immunological properties will allow a better understanding of their role in cellular therapy. PMID:24590129

  2. Liquid contact resonance AFM: analytical models, experiments, and limitations

    NASA Astrophysics Data System (ADS)

    Parlak, Zehra; Tu, Qing; Zauscher, Stefan

    2014-11-01

    Contact resonance AFM (CR-AFM) is a scanning probe microscopy technique that utilizes the contact resonances of the AFM cantilever for concurrent imaging of topography and surface stiffness. The technique has not been used in liquid until recently due to analytical and experimental difficulties, associated with viscous damping of cantilever vibrations and fluid loading effects. To address these difficulties, (i) an analytical approach for contact resonances in liquid is developed, and (ii) direct excitation of the contact resonances is demonstrated by actuating the cantilever directly in a magnetic field. By implementing the analytical approach and the direct actuation through magnetic particles, quantitative stiffness imaging on surfaces with a wide range of stiffness can be achieved in liquid with soft cantilevers and low contact forces.

  3. Ability of Lactobacillus rhamnosus GAF01 to remove AFM1 in vitro and to counteract AFM1 immunotoxicity in vivo.

    PubMed

    Abbès, Samir; Salah-Abbès, Jalila Ben; Sharafi, Hakimeh; Jebali, Rania; Noghabi, Kambiz Akbari; Oueslati, Ridha

    2013-01-01

    Aflatoxin M1 (AFM1) has been detected in many parts of the world both in raw milk and many dairy products, causing great economic losses and human disease. Unfortunately, there are few studies dealing with AFM1 immunotoxicity/interactions with lactic acid bacteria for potential application as a natural preventive agent. The aim of this study was to isolate (from dairy products) food-grade probiotic bacteria able to degrade/bind AFM1 in vitro and evaluate whether the same organism(s) could impart a protective role against AFM1-induced immunotoxicity in exposed Balb/c mice. Bacteria (Lactobacillus plantarum MON03 and L. rhamnosus GAF01) were isolated from Tunisian artisanal butter and then tested for abilities to eliminate AFM1 from phosphate-buffered saline (PBS) and reconstituted milk (containing 0.05, 0.10, and 0.20 µg AFM1/ml) after 0, 6, and 24 h at 37°C. Results showed that the selected bacteria could 'remove' AFM1 both in PBS and skimmed milk. The binding abilities of AFM1 by L. plantarum MON03 and L. rhamnosus GAF01 strains (at 10(8) CFU/ml) in PBS and reconstituted milk ranged, respectively, from 16.1-78.6% and 15.3-95.1%; overall, L. rhamnosus showed a better potential for removal than L. plantarum. 'Removal' appeared to be by simple binding; the bacteria/AFM1 complex was stable and only a very small proportion of mycotoxin was released back into the solution. L. rhamnosus GAF01 had the highest binding capacity and was selected for use in the in vivo study. Those results indicated that use of the organism prevented AFM1-induced effects on total white and red blood cells, and lymphocyte subtypes, after 15 days of host treatment. These studies clearly indicated that L. rhamnosus GAF01 was able to bind AFM1 in vitro and-by mechanisms that might also be related to a binding effect-counteract AFM1-induced immunotoxicity. Moreover, by itself, this bacterium was not toxic and could potentially be used as an additive in dairy products and in biotechnology for

  4. Fluid models and simulations of biological cell phenomena

    NASA Technical Reports Server (NTRS)

    Greenspan, H. P.

    1982-01-01

    The dynamics of coated droplets are examined within the context of biofluids. Of specific interest is the manner in which the shape of a droplet, the motion within it as well as that of aggregates of droplets can be controlled by the modulation of surface properties and the extent to which such fluid phenomena are an intrinsic part of cellular processes. From the standpoint of biology, an objective is to elucidate some of the general dynamical features that affect the disposition of an entire cell, cell colonies and tissues. Conventionally averaged field variables of continuum mechanics are used to describe the overall global effects which result from the myriad of small scale molecular interactions. An attempt is made to establish cause and effect relationships from correct dynamical laws of motion rather than by what may have been unnecessary invocation of metabolic or life processes. Several topics are discussed where there are strong analogies droplets and cells including: encapsulated droplets/cell membranes; droplet shape/cell shape; adhesion and spread of a droplet/cell motility and adhesion; and oams and multiphase flows/cell aggregates and tissues. Evidence is presented to show that certain concepts of continuum theory such as suface tension, surface free energy, contact angle, bending moments, etc. are relevant and applicable to the study of cell biology.

  5. Coupling EELS/EFTEM Imaging with Environmental Fluid Cell Microscopy

    SciTech Connect

    Unocic, Raymond R; Baggetto, Loic; Veith, Gabriel M; Dudney, Nancy J; More, Karren Leslie

    2012-01-01

    Insight into dynamically evolving electrochemical reactions and mechanisms encountered in electrical energy storage (EES) and conversion technologies (batteries, fuel cells, and supercapacitors), materials science (corrosion and oxidation), and materials synthesis (electrodeposition) remains limited due to the present lack of in situ high-resolution characterization methodologies. Electrochemical fluid cell microscopy is an emerging in-situ method that allows for the direct, real-time imaging of electrochemical processes within a fluid environment. This technique is facilitated by the use of MEMS-based biasing microchip platforms that serve the purpose of sealing the highly volatile electrolyte between two electron transparent SiNx membranes and interfacing electrodes to an external potentiostat for controlled nanoscale electrochemislly experiments [!]. In order to elucidate both stmctural and chemical changes during such in situ electrochemical experiments, it is impmtant to first improve upon the spatial resolution by utilizing energy-filtered transmission electron microscopy (EFTEM) (to minimize chromatic aben ation), then to detennine the chemical changes via electron energy loss spectroscopy (EELS). This presents a formidable challenge since the overall thickness through which electrons are scattered through the multiple layers of the cell can be on the order of hundreds of nanometers to microns, scattering through which has the deleterious effect of degrading image resolution and decreasing signal-to noise for spectroscopy [2].

  6. Confocal Raman microscopy of pathologic cells in cerebrospinal fluid

    NASA Astrophysics Data System (ADS)

    Gonchukov, S. A.; Lonkina, T. V.; Minaeva, S. A.; Sundukov, A. V.; Migmanov, T. E.; Lademann, J.; Darvin, M. E.; Bagratashvili, V. N.

    2014-01-01

    In this work, the spatial localization of leucocytes, bacteria, and erythrocytes in the crystal pattern of a dried droplet of cerebrospinal fluid (CSF) is established. Characteristic lines are detected and identified in the Raman spectrum of the CSF that point to the presence of pathologic cells therein and can be used in a timely way to diagnose meningitis, the spectroscopic sample preparation procedure being simple enough. A dry CSF sample retains its characteristic spectral features for no less than three days, which is important for its safe keeping and transportation, and also for the computer processing of its spectra.

  7. Lymphatic vessel development: fluid flow and valve-forming cells.

    PubMed

    Kume, Tsutomu

    2015-08-01

    Hemodynamic forces regulate many aspects of blood vessel disease and development, including susceptibility to atherosclerosis and remodeling of primary blood vessels into a mature vascular network. Vessels of the lymphatic circulatory system are also subjected to fluid flow-associated forces, but the molecular and cellular mechanisms by which these forces regulate the formation and maintenance of lymphatic vessels remain largely uncharacterized. This issue of the JCI includes two articles that begin to address how fluid flow influences lymphatic vessel development and function. Sweet et al. demonstrate that lymph flow is essential for the remodeling of primary lymphatic vessels, for ensuring the proper distribution of smooth muscle cells (SMCs), and for the development and maturation of lymphatic valves. Kazenwadel et al. show that flow-induced lymphatic valve development is initiated by the upregulation of GATA2, which has been linked to lymphedema in patients with Emberger syndrome. Together, these observations and future studies inspired by these results have potential to lead to the development of strategies for the treatment of lymphatic disorders. PMID:26214518

  8. Mounting of Escherichia coli spheroplasts for AFM imaging.

    SciTech Connect

    Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P; Doktycz, Mitchel John

    2005-11-01

    The cytoplasmic membrane of Escherichia coli (E. coli) is the location of numerous, chemically specific transporters and recognition elements. Investigation of this membrane in vivo by atomic force microscopy (AFM) requires removal of the cell wall and stable immobilization of the spheroplast. AFM images demonstrate that spheroplasts can be secured with warm gelatin applied to the mica substrate just before the addition of a spheroplast suspension. The resulting preparation can be repeatedly imaged by AFM over the course of several hours. Confocal fluorescence imaging confirms the association of the spheroplasts with the gelatin layer. Gelatin molecules are known to reorder into a network after heating. Entrapment within this gelatin network is believed to be responsible for the immobilization of spheroplasts on mica.

  9. A Mini Overview of Isolation, Characterization and Application of Amniotic Fluid Stem Cells.

    PubMed

    Gholizadeh-Ghalehaziz, Shiva; Farahzadi, Raheleh; Fathi, Ezzatollah; Pashaiasl, Maryam

    2015-11-01

    Amniotic fluid represents rich sources of stem cells that can be used in treatments for a wide range of diseases. Amniotic fluid- stem cells have properties intermediate between embryonic and adult mesenchymal stem cells which make them particularly attractive for cellular regeneration and tissue engineering. Furthermore, scientists are interested in these cells because they come from the amniotic fluid that is routinely discarded after birth. In this review we give a brief introduction of amniotic fluid followed by a description of the cells present within this fluid and aim to summarize the all existing isolation methods, culturing, characterization and application of these cells. Finally, we elaborate on the differentiation and potential for these cells to promote regeneration of various tissue defects, including fetal tissue, the nervous system, heart, lungs, kidneys, bones, and cartilage in the form of table. PMID:26634059

  10. Role of cells in freezing-induced cell-fluid-matrix interactions within engineered tissues.

    PubMed

    Seawright, Angela; Ozcelikkale, Altug; Dutton, Craig; Han, Bumsoo

    2013-09-01

    During cryopreservation, ice forms in the extracellular space resulting in freezing-induced deformation of the tissue, which can be detrimental to the extracellular matrix (ECM) microstructure. Meanwhile, cells dehydrate through an osmotically driven process as the intracellular water is transported to the extracellular space, increasing the volume of fluid for freezing. Therefore, this study examines the effects of cellular presence on tissue deformation and investigates the significance of intracellular water transport and cell-ECM interactions in freezing-induced cell-fluid-matrix interactions. Freezing-induced deformation characteristics were examined through cell image deformetry (CID) measurements of collagenous engineered tissues embedded with different concentrations of MCF7 breast cancer cells versus microspheres as their osmotically inactive counterparts. Additionally, the development of a biophysical model relates the freezing-induced expansion of the tissue due to the cellular water transport and the extracellular freezing thermodynamics for further verification. The magnitude of the freezing-induced dilatation was found to be not affected by the cellular water transport for the cell concentrations considered; however, the deformation patterns for different cell concentrations were different suggesting that cell-matrix interactions may have an effect. It was, therefore, determined that intracellular water transport during freezing was insignificant at the current experimental cell concentrations; however, it may be significant at concentrations similar to native tissue. Finally, the cell-matrix interactions provided mechanical support on the ECM to minimize the expansion regions in the tissues during freezing. PMID:23719856

  11. Microrheology using a custom-made AFM

    NASA Astrophysics Data System (ADS)

    Kosgodagan Acharige, Sebastien; Benzaquen, Michael; Steinberger, Audrey

    In the past few years, a new method was developed to measure local properties of liquids (X. Xiong et al., Phys. Rev. E 80, 2009). This method consists of gluing a micron-sized glass fiber at the tip of an AFM cantilever and probing the liquid with it. In ENS Lyon, this method was perfected (C. Devailly et al., EPL, 106 5, 2014) with the help of an interferometer developped in the same laboratory (L. Bellon et al., Opt. Commun. 207 49, 2002 and P. Paolino et al., Rev. Sci. Instrum. 84, 2013), which background noise can reach 10-14 m /√{ Hz } . This method allows us to measure a wide range of viscosities (1 mPa . s to 500 mPa . s) of transparent and opaque fluids using a small sample volume ( 5 mL). In this presentation, I will briefly describe the interferometer developped in ENS Lyon, then explain precisely the microrheology measurements and then compare the experimental results to a model developped by M. Benzaquen. This work is supported financially by the ANR project NANOFLUIDYN (Grant Number ANR-13-BS10-0009).

  12. Manufacturing process of nanofluidics using afm probe

    NASA Astrophysics Data System (ADS)

    Karingula, Varun Kumar

    A new process for fabricating a nano fluidic device that can be used in medical application is developed and demonstrated. Nano channels are fabricated using a nano tip in indentation mode on AFM (Atomic Force Microscopy). The nano channels are integrated between the micro channels and act as a filter to separate biomolecules. Nano channels of 4 to7 m in length, 80nm in width, and at varying depths from 100nm to 850 nm allow the resulting device to separate selected groups of lysosomes and other viruses. Sharply developed vertical micro channels are produced from a deep reaction ion etching followed by deposition of different materials, such as gold and polymers, on the top surface, allowing the study of alternative ways of manufacturing a nanofluidic device. PDMS (Polydimethylsiloxane) bonding is performed to close the top surface of the device. An experimental setup is used to test and validate the device by pouring fluid through the channels. A detailed cost evaluation is conducted to compare the economical merits of the proposed process. It is shown that there is a 47:7% manufacturing time savings and a 60:6% manufacturing cost savings.

  13. Use of the Cell-Dyn Sapphire hematology analyzer for automated counting of blood cells in body fluids.

    PubMed

    De Smet, Dieter; Van Moer, Guy; Martens, Geert A; Nanos, Nikolaos; Smet, Lutgarde; Jochmans, Kristin; De Waele, Marc

    2010-02-01

    The enumeration and identification of blood cells in body fluids offers important information for the diagnosis and treatment of various medical conditions. Manual microscopic methods (hemacytometer total cell count and cytocentrifuged differential count) have inherent analytic and economic disadvantages but are still considered the "gold standard" methods. We evaluated the analytic and clinical performance of the Cell-Dyn Sapphire hematology analyzer (Abbott Diagnostics Division, Santa Clara, CA) for automated blood cell counting and leukocyte differential counting in cerebrospinal fluid, serous fluid (peritoneal and pleural fluid), and continuous ambulatory peritoneal dialysis fluid, and we compared the performance with the respective manual methods. In the present article, we describe its applicability for the distinct body fluids, and we highlight limitations and caveats. PMID:20093239

  14. Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells.

    PubMed

    Wang, Han Chin; Lin, Chun-Chieh; Cheung, Rocky; Zhang-Hooks, YingXin; Agarwal, Amit; Ellis-Davies, Graham; Rock, Jason; Bergles, Dwight E

    2015-12-01

    Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl(-) efflux and osmotic cell shrinkage by opening TMEM16A Ca(2+)-activated Cl(-) channels. Release of Cl(-) from ISCs also forces K(+) efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea and prevents ATP-dependent shrinkage of supporting cells. These results indicate that supporting cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells. PMID:26627734

  15. Single ricin detection by AFM chemomechanical mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research reports a method of detecting ricin molecules immobilized on chemically modified gold (Au;111) surface by chemomechanically mapping the molecular interactions with a chemically modified Atomic Force Microscope (AFM) tip. AFM images resolved the different fold-up conformations of single...

  16. Satellited 4q identified in amniotic fluid cells

    SciTech Connect

    Miller, I.; Hsieh, C.L.; Songster, G.

    1995-01-16

    Extra material was identified on the distal long arm of a chromosome 4 in an amniotic fluid specimen sampled at 16.6 weeks of gestational age. There was no visible loss of material from chromosome 4, and no evidence for a balanced rearrangement. The primary counseling issue in this case was advanced maternal age. Ultrasound findings were normal, and family history was unremarkable. The identical 4qs chromosome was observed in cells from a paternal peripheral blood specimen and appeared to be an unbalanced rearrangement. This extra material was NOR positive in lymphocytes from the father, but was negative in the fetal amniocytes. Father`s relatives were studied to verify the familial origin of this anomaly. In situ hybridization with both exon and intron sequences of ribosomal DNA demonstrated that ribosomal DNA is present at the terminus of the 4qs chromosome in the fetus, father, and paternal grandmother. This satellited 4q might have been derived from a translocation event that resulted in very little or no loss from the 4q and no specific phenotype. This derivative chromosome 4 has been inherited through at least 3 generations of phenotypically normal individuals. 8 refs., 3 figs.

  17. Rabbit tendon cells produce MMP-3 in response to fluid flow without significant calcium transients.

    PubMed

    Archambault, Joanne M; Elfervig-Wall, Michelle K; Tsuzaki, Mari; Herzog, Walter; Banes, Albert J

    2002-03-01

    Forces applied to tendon during movement cause cellular deformation, as well as fluid movement. The goal of this study was to test the hypothesis that rabbit tendon fibroblasts detect and respond to fluid-induced shear stress. Cells were isolated from the paratenon of the rabbit Achilles tendon and then subjected to fluid flow at 1 dyn/cm(2) for 6h in a specially designed multi-slide flow device. The application of fluid flow led to an increased expression of the collagenase-1 (MMP-1), stromelysin-1 (MMP-3), cyclooxygenase II (COX-2) and interleukin-1beta (IL-1beta) genes. The release of proMMP-3 into the medium exhibited a dose-response with the level of fluid shear stress. However, not all cells aligned in the direction of flow. In other experiments, the same cells were incubated with the calcium-reactive dye FURA-2 AM, then subjected to laminar fluid flow in a parallel plate flow chamber. The cells did not significantly increase intracellular calcium concentration when exposed to fluid shear stress levels of up to 25 dyn/cm(2). These results show that gene expression in rabbit tendon cells is sensitive to fluid flow, but that signal transduction is not dependent on intracellular calcium transients. The upregulation of the MMP-1, MMP-3 and COX-2 genes shows that fluid flow could be an important mechanical stimulus for tendon remodelling or injury. PMID:11858805

  18. Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

    PubMed

    Soltani, M; Nemati, M; Maralani, M; Estiar, M A; Andalib, S; Fardiazar, Z; Sakhinia, E

    2016-01-01

    In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of β-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common β-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and β-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis. PMID:27188728

  19. Interstitial fluid flow: simulation of mechanical environment of cells in the interosseous membrane

    NASA Astrophysics Data System (ADS)

    Yao, Wei; Ding, Guang-Hong

    2011-08-01

    In vitro experiments have shown that subtle fluid flow environment plays a significant role in living biological tissues, while there is no in vivo practical dynamical measurement of the interstitial fluid flow velocity. On the basis of a new finding that capillaries and collagen fibrils in the interosseous membrane form a parallel array, we set up a porous media model simulating the flow field with FLUENT software, studied the shear stress on interstitial cells' surface due to the interstitial fluid flow, and analyzed the effect of flow on protein space distribution around the cells. The numerical simulation results show that the parallel nature of capillaries could lead to directional interstitial fluid flow in the direction of capillaries. Interstitial fluid flow would induce shear stress on the membrane of interstitial cells, up to 30 Pa or so, which reaches or exceeds the threshold values of cells' biological response observed in vitro. Interstitial fluid flow would induce nonuniform spacial distribution of secretion protein of mast cells. Shear tress on cells could be affected by capillary parameters such as the distance between the adjacent capillaries, blood pressure and the permeability coefficient of capillary's wall. The interstitial pressure and the interstitial porosity could also affect the shear stress on cells. In conclusion, numerical simulation provides an effective way for in vivo dynamic interstitial velocity research, helps to set up the vivid subtle interstitial flow environment of cells, and is beneficial to understanding the physiological functions of interstitial fluid flow.

  20. Investigation of the Mechanoelectrical Transduction at Single Stereocilia by Afm

    NASA Astrophysics Data System (ADS)

    Langer, M. G.; Fink, S.; Löffler, K.; Koitschev, A.; Zenner, H.-P.

    2003-02-01

    The transduction of sound into an electrical signal in the inner ear is closely related to the mechanical properties of the hair bundles cytoskeleton and cross-linkage. In this study the effect of lateral cross-links on hair bundle mechanics and the transduction current response is demonstrated on the level of individual stereocilia. For experiments stereocilia of outer hair cells of postnatal rats (P3 - P8) were scanned with a sharp AFM tip at nanometerscale. Transduction currents were simultaneously recorded in the whole-cell-recording mode with patch clamp. AFM was used as a nanotool for local mechanical stimulation and force measurement at stereocilia whereas patch clamp serves as a detector for the electrical response of the cell. In a first experiment force transmission between adjacent stereocilia of the V- and W- shaped hair bundles of outer hair cells was investigated. Results showed that a force exerted to a single stereocilium declined to 36 % at the nearest adjacent stereocilium of the same row. This result supposes AFM to be convenient for local displacement of single stereocilia. For control, the local response of transduction channels was measured at single stereocilia of the same hair bundle. Measured transduction current amplitudes ranged from 9 to 49 pA supposing an opening of one to five transduction channels. Both, weak force transmission by lateral cross-links and small transduction current amplitudes indicate a weak mechanical interaction between individual stereocilia of the tallest row of stereocilia of outer hair cells from postnatal rats.

  1. Introduction to Atomic Force Microscopy (AFM) in Biology.

    PubMed

    Kreplak, Laurent

    2016-01-01

    The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution over a wide range of time scales from milliseconds to hours. In addition to providing topographical images of surfaces with nanometer- to angstrom-scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated from the nano-scale to the micro-scale. Importantly, the measurements are made in buffer solutions, allowing biological samples to "stay alive" within a physiological-like environment while temporal changes in structure are measured-e.g., before and after addition of chemical reagents. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, e.g., electron microscopy (EM). This unit provides an introduction to AFM on biological systems and describes specific examples of AFM on proteins, cells, and tissues. The physical principles of the technique and methodological aspects of its practical use and applications are also described. © 2016 by John Wiley & Sons, Inc. PMID:27479503

  2. AFM imaging of functionalized carbon nanotubes on biological membranes

    NASA Astrophysics Data System (ADS)

    Lamprecht, C.; Liashkovich, I.; Neves, V.; Danzberger, J.; Heister, E.; Rangl, M.; Coley, H. M.; McFadden, J.; Flahaut, E.; Gruber, H. J.; Hinterdorfer, P.; Kienberger, F.; Ebner, A.

    2009-10-01

    Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

  3. ezAFM: A low cost Atomic Force Microscope(AFM)

    NASA Astrophysics Data System (ADS)

    Celik, Umit; Celik, Kubra; Aslan, Husnu; Kehribar, Ihsan; Dede, Munir; Ozgur Ozer, H.; Oral, Ahmet

    2012-02-01

    A low cost AFM, ezAFM is developed for educational purposes as well as research. Optical beam deflection method is used to measure the deflection of cantilever. ezAFM scanner is built using voice coil motors (VCM) with ˜50x50x6 μm scan area. The microscope uses alignment free cantilevers, which minimizes setup times. FPGA based AFM feedback Control electronics is developed. FPGA technology allows us to drive all peripherals in parallel. ezAFM Controller is connected to PC by USB 2.0 interface as well as Wi-Fi. We have achieved <5nm lateral and ˜0.01nm vertical resolution. ezAFM can image single atomic steps in HOPG and mica. An optical microscope with <3 μm resolution is also integrated into the system. ezAFM supports different AFM operation modes such as dynamic mode, contact mode, lateral force microscopy. Advanced modes like magnetic force microscopy and electric force microscopy will be implemented later on. The new ezAFM system provides, short learning times for student labs, quick setup and easy to transport for portable applications with the best price/performance ratio. The cost of the system starts from 15,000, with system performance comparable with the traditional AFM systems.

  4. A dynamic pressure view cell for acoustic stimulation of fluids--Micro-bubble generation and fluid movement in porous media.

    PubMed

    Stewart, Robert A; Shaw, J M

    2015-09-01

    The development and baseline operation of an acoustic view cell for observing fluids, and fluid-fluid and fluid-solid interfaces in porous media over the frequency range of 10-5000 Hz is described. This range includes the industrially relevant frequency range 500-5000 Hz that is not covered by existing devices. Pressure waveforms of arbitrary shape are generated in a 17.46 mm ID by 200 mm and 690.5 mm long glass tubes at flow rates up to 200 ml/min using a syringe pump. Peak-to-peak amplitudes exceeding 80 kPa are readily realized at frequencies from 10 to 5000 Hz in bubble free fluids when actuated with 20 Vpp as exemplified using castor oil. At resonant frequencies, peak-to-peak pressure amplitudes exceeding 500 kPa were obtained (castor oil at 2100 Hz when actuated with 20 Vpp). Impacts of vibration on macroscopic liquid-liquid and liquid-vapour interfaces and interface movement are illustrated. Pressure wave transmission and attenuation in a fluid saturated porous medium, randomly packed 250-330 μm spherical silica beads, is also demonstrated. Attenuation differences and frequency shifts in resonant peaks are used to detect the presence and generation of dispersed micro-bubbles (<180 μm diameter), and bubbles within porous media that are not readily visualized. Envisioned applications include assessment of the impacts of vibration on reaction, mass transfer, and flow/flow pattern outcomes. This knowledge will inform laboratory and pilot scale process studies, where nuisance vibrations may affect the interpretation of process outcomes, and large scale or in situ processes in aquifers or hydrocarbon reservoirs where imposed vibration may be deployed to improve aspects of process performance. Future work will include miscible interface observation and quantitative measurements in the bulk and in porous media where the roles of micro-bubbles comprise subjects of special interest. PMID:26429474

  5. Studies of cell toxicity of complexes of magnetic fluids and biological macromolecules

    NASA Astrophysics Data System (ADS)

    Macaroff, Patrícia P.; Oliveira, Daniela M.; Ribeiro, Karina F.; Lacava, Zulmira G. M.; Lima, Emília C. D.; Morais, Paulo C.; Tedesco, Antonio C.

    2005-05-01

    In this study, we performed a comparative investigation of the binding properties of two surface-coated (carboxymethyldextran/glucuronic acid), magnetite-based biocompatible magnetic fluids with different biological macromolecules (BSA, HSA, and LDL). We also investigated the in vitro toxicity of the complex formed between the magnetic fluid and the biological macromolecule in the neoplastic cell line J774-A.

  6. Reusable, compression-sealed fluid cells for surface mounting to planar substrates.

    PubMed

    Tamanaha, Cy R; Malito, Michael P; Mulvaney, Shawn P; Whitman, Lloyd J

    2009-05-21

    We have developed a universal structure and mechanism for the repeatable, rapid-attachment of a fluid cell to a planar substrate. The fluid cell and all fluidic connections are completely contained in a plastic body such that attachment requires neither adhesives nor modification of the substrate. The geometry of the fluid cell is defined by the active area of the planar substrate (e.g. a sensor array). All required components have been quickly prototyped using Computer Numerical Control (CNC) machining. It is also straight-forward to create an array of fluid cells to attach to a single substrate (e.g. a standard microscope slide). All components are easy to assemble and can be cleaned and reused, making this flexible approach applicable for a wide range of lab-on-a-chip applications. PMID:19417916

  7. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    PubMed Central

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-01-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes. PMID:25778677

  8. Flagellar kinematics and swimming of algal cells in viscoelastic fluids.

    PubMed

    Qin, B; Gopinath, A; Yang, J; Gollub, J P; Arratia, P E

    2015-01-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes. PMID:25778677

  9. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    NASA Astrophysics Data System (ADS)

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-03-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes.

  10. Fluid and cell behaviors along a 3D printed alginate/gelatin/fibrin channel.

    PubMed

    Xu, Yufan; Wang, Xiaohong

    2015-08-01

    Three-dimensional (3D) cell manipulation is available with the integration of microfluidic technology and rapid prototyping techniques. High-Fidelity (Hi-Fi) constructs hold enormous therapeutic potential for organ manufacturing and regenerative medicine. In the present paper we introduced a quasi-three-dimensional (Q3D) model with parallel biocompatible alginate/gelatin/fibrin hurdles. The behaviors of fluids and cells along the microfluidic channels with various widths were studied. Cells inside the newly designed microfluidic channels attached and grew well. Morphological changes of adipose-derived stem cells (ADSCs) in both two-dimensional (2D) and 3D milieu were found on the printed constructs. Endothelialization occurred with the co-cultures of ADSCs and hepatocytes. This study provides insights into the interactions among fluids, cells and biomaterials, the behaviors of fluids and cells along the microfluidic channels, and the applications of Q3D techniques. PMID:25727058

  11. Nano Mechanical Machining Using AFM Probe

    NASA Astrophysics Data System (ADS)

    Mostofa, Md. Golam

    Complex miniaturized components with high form accuracy will play key roles in the future development of many products, as they provide portability, disposability, lower material consumption in production, low power consumption during operation, lower sample requirements for testing, and higher heat transfer due to their very high surface-to-volume ratio. Given the high market demand for such micro and nano featured components, different manufacturing methods have been developed for their fabrication. Some of the common technologies in micro/nano fabrication are photolithography, electron beam lithography, X-ray lithography and other semiconductor processing techniques. Although these methods are capable of fabricating micro/nano structures with a resolution of less than a few nanometers, some of the shortcomings associated with these methods, such as high production costs for customized products, limited material choices, necessitate the development of other fabricating techniques. Micro/nano mechanical machining, such an atomic force microscope (AFM) probe based nano fabrication, has, therefore, been used to overcome some the major restrictions of the traditional processes. This technique removes material from the workpiece by engaging micro/nano size cutting tool (i.e. AFM probe) and is applicable on a wider range of materials compared to the photolithographic process. In spite of the unique benefits of nano mechanical machining, there are also some challenges with this technique, since the scale is reduced, such as size effects, burr formations, chip adhesions, fragility of tools and tool wear. Moreover, AFM based machining does not have any rotational movement, which makes fabrication of 3D features more difficult. Thus, vibration-assisted machining is introduced into AFM probe based nano mechanical machining to overcome the limitations associated with the conventional AFM probe based scratching method. Vibration-assisted machining reduced the cutting forces

  12. [LE cells in synovial fluid: prevalence and diagnostic usefulness in rheumatic diseases].

    PubMed

    Puszczewicz, Mariusz; Białkowska-Puszczewicz, Grazyna

    2010-01-01

    This study was undertaken to determine the prevalence of LE cells in synovial fluid and their importance for the diagnosis of rheumatic disease. Synovial fluid was obtained from 631 patients: 31 with systemic lupus erythematosus (SLE), 337 with rheumatoid arthritis (RA), 4 with Still's disease, 9 with systemic scleroderma (SS), 27 with the overlap syndrome (RA/SLE), 132 with ankylosing spondylitis (AS), 57 with Reiter's syndrome, and 34 with psoriatic arthritis (PA). The fluid was centrifuged, precipitate smears were done and were May-Grünwald-Giemsa stained for cytologic assessment. The supernatant was collected for antinuclear antibody (ANA) testing. Physicochemical and serologic properties of the synovial fluid were routinely determined. All synovial fluids demonstrated signs of inflammation. The presence of LE cells was ascertained in five patients with SLE and nine patients with the overlap syndrome. In these cases, LE cells were accompanied by ANA. In addition, hematoxylin bodies were revealed in SLE patients. LE cells were observed in 2.6% of patients with RA but were not accompanied by ANA. Patients with SS, Still's disease, AS, Reiter's syndrome, and PA tested negative for LE cells. It appears from these results that LE cells are rarely present in the synovial fluid of patients with rheumatic diseases. In contrast, they occur in more than 40% of patients with the overlap syndrome and may thus be regarded as important for the diagnosis of this condition. PMID:21365954

  13. Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

    PubMed Central

    Miltenburg, A M; Van Laar, J M; De Kuiper, P; Daha, M R; Breedveld, F C

    1990-01-01

    A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function. PMID:2148285

  14. Method for filling the cavities of cells with a chromogenic fluid

    SciTech Connect

    Tonazzi, J.C.L.; Kucharczyk, J.E. Jr.; Agrawal, A.

    1999-01-05

    A method and apparatus are disclosed for filling a cell cavity positioned between a first substrate and a second substrate with a cell filling liquid. The method entails forming at least one evacuation cavity encompassing at least a portion of an outer surface of each of the first and second substrates of a cell containing a cell cavity and isolating the cell cavity from the evacuation cavity; reducing a pressure in each of the evacuation cavity and the cell cavity; and dispensing the cell filling fluid into the cell cavity. The application is to the fabrication of electrochromic windows. 22 figs.

  15. Method for filling the cavities of cells with a chromogenic fluid

    DOEpatents

    Tonazzi, Juan C. Lopez; Kucharczyk, Jr., Joseph E.; Agrawal, Anoop

    1999-01-01

    A method and apparatus are disclosed for filling a cell cavity positioned between a first substrate and a second substrate with a cell filling liquid. The method entails forming at least one evacuation cavity encompassing at least a portion of an outer surface of each of the first and second substrates of a cell containing a cell cavity and isolating the cell cavity from the evacuation cavity; reducing a pressure in each of the evacuation cavity and the cell cavity; and dispensing the cell filling fluid into the cell cavity.

  16. Apparatus for filling the cavities of cells and laminated substrates with a fluid

    DOEpatents

    Lopez Tonazzi, Juan C.; Kucharczyk, Jr., Joseph E.; Agrawal, Anoop

    2001-01-01

    A method and apparatus are disclosed for filling a cell cavity positioned between a first substrate and a second substrate with a cell filling liquid. The method entails forming at least one evacuation cavity encompassing at least a portion of an outer surface of each of the first and second substrates of a cell containing a cell cavity and isolating the cell cavity from the evacuation cavity; reducing a pressure in each of the evacuation cavity and the cell cavity; and dispensing the cell filling fluid into the cell cavity.

  17. Reprogramming of mouse amniotic fluid cells using a PiggyBac transposon system.

    PubMed

    Bertin, E; Piccoli, M; Franzin, C; Nagy, A; Mileikovsky, M; De Coppi, P; Pozzobon, M

    2015-11-01

    Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Amniotic fluid (AF) cells are easy to obtain from routinely scheduled procedures for prenatal diagnosis and iPS cells have been generated from human AF. Here, we generated iPS cells from mouse AF cells, using a non-viral-based approach constituted by the PiggyBac (PB) transposon system. All iPS cell lines obtained exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. PMID:26987920

  18. Membrane-based actuation for high-speed single molecule force spectroscopy studies using AFM.

    PubMed

    Sarangapani, Krishna; Torun, Hamdi; Finkler, Ofer; Zhu, Cheng; Degertekin, Levent

    2010-07-01

    Atomic force microscopy (AFM)-based dynamic force spectroscopy of single molecular interactions involves characterizing unbinding/unfolding force distributions over a range of pulling speeds. Owing to their size and stiffness, AFM cantilevers are adversely affected by hydrodynamic forces, especially at pulling speeds >10 microm/s, when the viscous drag becomes comparable to the unbinding/unfolding forces. To circumvent these adverse effects, we have fabricated polymer-based membranes capable of actuating commercial AFM cantilevers at speeds >or=100 microm/s with minimal viscous drag effects. We have used FLUENT, a computational fluid dynamics (CFD) software, to simulate high-speed pulling and fast actuation of AFM cantilevers and membranes in different experimental configurations. The simulation results support the experimental findings on a variety of commercial AFM cantilevers and predict significant reduction in drag forces when membrane actuators are used. Unbinding force experiments involving human antibodies using these membranes demonstrate that it is possible to achieve bond loading rates >or=10(6) pN/s, an order of magnitude greater than that reported with commercial AFM cantilevers and systems. PMID:20054686

  19. Emergent long-range couplings in arrays of fluid cells

    SciTech Connect

    Abraham, Douglas Bruce

    2014-08-07

    We present a system exhibiting extraordinarily long-range cooperative effects, on a length scale far exceeding the bulk correlation length. We give a theoretical explanation of these phenomena based on the mesoscopic picture of phase coexistence in finite systems, which is confirmedly Monte Carlo (MC) simulation studies. Our work demonstrates that such action-at-a-distance can occur in classical systems involving simple or complex fluids, such as colloid-polymer mixtures, or ferromagnets.

  20. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential.

    PubMed

    Pipino, Caterina; Pandolfi, Assunta

    2015-05-26

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine. PMID:26029340

  1. Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

    PubMed Central

    Pipino, Caterina; Pandolfi, Assunta

    2015-01-01

    In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine. PMID:26029340

  2. Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains

    PubMed Central

    Minev, Boris R.; Zimmermann, Martina; Aguilar, Richard J.; Zhang, Qian; Sturm, Julia B.; Fend, Falko; Yu, Yong A.; Cappello, Joseph; Lauer, Ulrich M.; Szalay, Aladar A.

    2013-01-01

    Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease. PMID:24019862

  3. Hydration states of AFm cement phases

    SciTech Connect

    Baquerizo, Luis G.; Matschei, Thomas; Scrivener, Karen L.; Saeidpour, Mahsa; Wadsö, Lars

    2015-07-15

    The AFm phase, one of the main products formed during the hydration of Portland and calcium aluminate cement based systems, belongs to the layered double hydrate (LDH) family having positively charged layers and water plus charge-balancing anions in the interlayer. It is known that these phases present different hydration states (i.e. varying water content) depending on the relative humidity (RH), temperature and anion type, which might be linked to volume changes (swelling and shrinkage). Unfortunately the stability conditions of these phases are insufficiently reported. This paper presents novel experimental results on the different hydration states of the most important AFm phases: monocarboaluminate, hemicarboaluminate, strätlingite, hydroxy-AFm and monosulfoaluminate, and the thermodynamic properties associated with changes in their water content during absorption/desorption. This data opens the possibility to model the response of cementitious systems during drying and wetting and to engineer systems more resistant to harsh external conditions.

  4. Electrokinetic Focusing and Separation of Mammalian Cells in Conductive Biological Fluids

    PubMed Central

    Gao, Jian Gao; Riahi, Reza; Sin, Mandy L. Y.; Zhang, Shufeng; Wong, Pak Kin

    2014-01-01

    Active manipulation of cells, such as trapping, focusing, and isolation, is essential for various bioanalytical applications. Herein, we report a hybrid electrokinetic technique for manipulating mammalian cells in physiological fluids. This technique applies a combination of negative dielectrophoretic force and hydrodynamic drag force induced by electrohydrodynamics, which is effective in conductive biological fluids. With a three-electrode configuration, the stable equilibrium positions of cells can be adjusted for separation and focusing applications. Cancer cells and white blood cells can be positioned and isolated into specific locations in the microchannel under both static and dynamic flow conditions. To investigate the sensitivity of the hybrid electrokinetic process, AC voltage, frequency, and bias dependences of the cell velocity were studied systematically. The applicability of the hybrid electrokinetic technique for manipulating cells in physiological samples is demonstrated by continuous focusing human breast adenocarcinoma spiked in urine, buffy coats, and processed blood samples with 98% capture efficiency. PMID:22937529

  5. Electrokinetic focusing and separation of mammalian cells in conductive biological fluids.

    PubMed

    Gao, Jian; Riahi, Reza; Sin, Mandy L Y; Zhang, Shufeng; Wong, Pak Kin

    2012-11-21

    Active manipulation of cells, such as trapping, focusing, and isolation, is essential for various bioanalytical applications. Herein, we report a hybrid electrokinetic technique for manipulating mammalian cells in physiological fluids. This technique applies a combination of negative dielectrophoretic force and hydrodynamic drag force induced by electrohydrodynamics, which is effective in conductive biological fluids. With a three-electrode configuration, the stable equilibrium positions of cells can be adjusted for separation and focusing applications. Cancer cells and white blood cells can be positioned and isolated into specific locations in the microchannel under both static and dynamic flow conditions. To investigate the sensitivity of the hybrid electrokinetic process, AC voltage, frequency, and bias dependences of the cell velocity were studied systematically. The applicability of the hybrid electrokinetic technique for manipulating cells in physiological samples is demonstrated by continuous focusing of human breast adenocarcinoma spiked in urine, buffy coats, and processed blood samples with 98% capture efficiency. PMID:22937529

  6. LET Spectrum Measurements In CR-39 PNTD With AFM

    NASA Astrophysics Data System (ADS)

    Johnson, C. E.; DeWitt, J. M.; Benton, E. R.; Yasuda, N.; Benton, E. V.

    2011-06-01

    Energetic protons, neutrons, and heavy ions undergoing collisions with target nuclei of varying Z can produce residual heavy recoil fragments via intra-nuclear cascade/evaporation reactions. The particles produced in these non-elastic collisions generally have such extremely short range (˜<10 μm) that they cannot be directly observed by conventional detection methods including CR-39 plastic nuclear track detector (PNTD) that has been chemically etched for analysis by standard visible light microscopy. However, high-LET recoil fragments having range on the order of several cell diameters can be produced in tissue during radiotherapy using proton and carbon beams. We have developed a method to analyze short-range, high-LET tracks in CR-39 plastic nuclear track detector (PNTD) using short duration chemical etching (˜<1 μm) following by automated atomic force microscope (AFM) scanning. The post-scan data processing used in this work was based on semi-automated matrix analysis opposed to traditional grey-scale image analysis. This method takes advantage of the 3-D data obtained via AFM to achieve robust discrimination of nuclear tracks from other features inherently present in the post-etch detector surface. Through automation of AFM scanning, sufficient AFM scan frames were obtained to attain an LET spectrum spanning the LET range from 200-1500 keV/μm. In addition to our experiments, simulations were carried out with the Monte Carlo transport code, FLUKA. To demonstrate this method, CR-39 PNTD was exposed to the proton therapy beam at Loma Linda University Medical Center (LLUMC) at 60 and 230 MeV. Additionally, detectors were exposed to 1 GeV protons at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). For these exposures CR-39 PNTD, Al and Cu target foils were used between detector layers.

  7. LET Spectrum Measurements In CR-39 PNTD With AFM

    SciTech Connect

    Johnson, C. E.; DeWitt, J. M.; Benton, E. R.; Yasuda, N.; Benton, E. V.

    2011-06-01

    Energetic protons, neutrons, and heavy ions undergoing collisions with target nuclei of varying Z can produce residual heavy recoil fragments via intra-nuclear cascade/evaporation reactions. The particles produced in these non-elastic collisions generally have such extremely short range ({approx}<10 {mu}m) that they cannot be directly observed by conventional detection methods including CR-39 plastic nuclear track detector (PNTD) that has been chemically etched for analysis by standard visible light microscopy. However, high-LET recoil fragments having range on the order of several cell diameters can be produced in tissue during radiotherapy using proton and carbon beams. We have developed a method to analyze short-range, high-LET tracks in CR-39 plastic nuclear track detector (PNTD) using short duration chemical etching ({approx}<1 {mu}m) following by automated atomic force microscope (AFM) scanning. The post-scan data processing used in this work was based on semi-automated matrix analysis opposed to traditional grey-scale image analysis. This method takes advantage of the 3-D data obtained via AFM to achieve robust discrimination of nuclear tracks from other features inherently present in the post-etch detector surface. Through automation of AFM scanning, sufficient AFM scan frames were obtained to attain an LET spectrum spanning the LET range from 200-1500 keV/{mu}m. In addition to our experiments, simulations were carried out with the Monte Carlo transport code, FLUKA. To demonstrate this method, CR-39 PNTD was exposed to the proton therapy beam at Loma Linda University Medical Center (LLUMC) at 60 and 230 MeV. Additionally, detectors were exposed to 1 GeV protons at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). For these exposures CR-39 PNTD, Al and Cu target foils were used between detector layers.

  8. LET spectrum measurements in Cr-39 PNTD with AFM

    SciTech Connect

    Johnson, Carl Edward; De Witt, Joel M; Benton, Eric R; Yasuda, Nakahiro; Benton, Eugene V

    2010-01-01

    Energetic protons, neutrons, and heavy ions undergoing collisions with target nuclei of varying Z can produce residual heavy recoil fragments via intra-nuclear cascade/evaporation reactions. The particles produced in these non-elastic collisions generally have such extremely short range ({approx}< 10 {mu}m) that they cannot be directly observed by conventional detection methods including CR-39 plastic nuclear track detector (PNTD) that has been chemically etched for analysis by standard visible light microscopy. However, high-LET recoil fragments having range on the order of several cell diameters can be produced in tissue during radiotherapy using proton and carbon beams. We have developed a method to analyze short-range, high-LET tracks in CR-39 plastic nuclear track detector (PNTD) using short duration chemical etching ({approx}< 1 {mu}m) followed by automated atomic force microscope (AFM) scanning. The post-scan data processing used in this work was based on semi-automated matrix analysis opposed to traditional grey-scale image analysis. This method takes advantage of the 3-D data obtained via AFM to achieve robust discrimination of nuclear tracks from other features. Through automation of AFM scanning, sufficient AFM scan frames were obtained to attain an LET spectrum spanning the LET range from 200-1500 keV/{mu}m. In addition to our experiments, simulations were carried out with the Monte Carlo transport code, FLUKA. To demonstrate this method, CR-39 PNTD was exposed to the proton therapy beam at Loma Linda University Medical Center (LLUMC) at 60 and 230 MeV. Additionally, detectors were exposed to I GeV protons at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). For these exposures CR-39 PNTD, Al and Cu target foils were used between detector layers.

  9. Relationship between automated total nucleated cell count and enumeration of cells on direct smears of canine synovial fluid

    PubMed Central

    Dusick, Allison; Young, Karen M.; Muir, Peter

    2016-01-01

    Canine osteoarthritis is a common condition seen in veterinary clinical practice and causes considerable morbidity in dogs as they age. Synovial fluid analysis is an important tool for diagnosis and treatment of canine joint disease and obtaining a total nucleated cell count (TNCC) is particularly important. The low volume of fluid obtained during arthrocentesis is often insufficient for obtaining an automated TNCC, thereby limiting sample interpretation. The aim of the present study was to investigate whether estimation of TNCC in canine synovial fluid could be achieved by performing manual cell counts on direct smears of fluid. Fifty eight synovial fluid samples, taken by arthrocentesis from 48 dogs, were included in the study. Direct smears of synovial fluid were prepared, and hyaluronidase added before cell counts were obtained using a commercial laser-based instrument. A protocol was established to count nucleated cells in a specific region of the smear, using a serpentine counting pattern; mean number of nucleated cells/400× field was then calculated. There was a positive correlation between the automated TNCC and mean manual cell count, with more variability at higher TNCC. Regression analysis was performed to estimate TNCC from manual counts. By this method, 78% of the samples were correctly predicted to fall into one of three categories (within the reference interval, mildly to moderately elevated, or markedly elevated) relative to the automated TNCC. Intra-observer and inter-observer agreement was good to excellent. The results of the study suggest that interpretation of canine synovial fluid samples of low volume can be aided by manual cell counting of direct smears. PMID:25439439

  10. A scanning acoustic microscope discriminates cancer cells in fluid

    NASA Astrophysics Data System (ADS)

    Miura, Katsutoshi; Yamamoto, Seiji

    2015-10-01

    Scanning acoustic microscopy (SAM) discriminates lesions in sections by assessing the speed of sound (SOS) or attenuation of sound (AOS) through tissues within a few minutes without staining; however, its clinical use in cytological diagnosis is unknown. We applied a thin layer preparation method to observe benign and malignant effusions using SAM. Although SAM is inferior in detecting nuclear features than light microscopy, it can differentiate malignant from benign cells using the higher SOS and AOS values and large irregular cell clusters that are typical features of carcinomas. Moreover, each single malignant cell exhibits characteristic cytoplasmic features such as a large size, irregular borders and secretory or cytoskeletal content. By adjusting the observation range, malignant cells are differentiated from benign cells easily using SAM. Subtle changes in the functional and structural heterogeneity of tumour cells were pursuable with a different digital data of SAM. SAM can be a useful tool for screening malignant cells in effusions before light microscopic observation. Higher AOS values in malignant cells compared with those of benign cells support the feasibility of a novel sonodynamic therapy for malignant effusions.

  11. Automated Static Culture System Cell Module Mixing Protocol and Computational Fluid Dynamics Analysis

    NASA Technical Reports Server (NTRS)

    Kleis, Stanley J.; Truong, Tuan; Goodwin, Thomas J,

    2004-01-01

    This report is a documentation of a fluid dynamic analysis of the proposed Automated Static Culture System (ASCS) cell module mixing protocol. The report consists of a review of some basic fluid dynamics principles appropriate for the mixing of a patch of high oxygen content media into the surrounding media which is initially depleted of oxygen, followed by a computational fluid dynamics (CFD) study of this process for the proposed protocol over a range of the governing parameters. The time histories of oxygen concentration distributions and mechanical shear levels generated are used to characterize the mixing process for different parameter values.

  12. Probing Cytoskeletal Structures by Coupling Optical Superresolution and AFM Techniques for a Correlative Approach

    PubMed Central

    Chacko, Jenu Varghese; Zanacchi, Francesca Cella; Diaspro, Alberto

    2013-01-01

    In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Young's modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution-AFM working is a clear example of correlative multimodal microscopy. PMID:24027190

  13. Conductance of AFM Deformed Carbon Nanotubes

    NASA Technical Reports Server (NTRS)

    Svizhenko, Alexei; Maiti, Amitesh; Anatram, M. P.; Biegel, Bryan (Technical Monitor)

    2002-01-01

    This viewgraph presentation provides information on the electrical conductivity of carbon nanotubes upon deformation by atomic force microscopy (AFM). The density of states and conductance were computed using four orbital tight-binding method with various parameterizations. Different chiralities develop bandgap that varies with chirality.

  14. A Batch Fabricated SECM-AFM Probe

    NASA Astrophysics Data System (ADS)

    Dobson, P. S.; Macpherson, J. V.; Holder, M.; Weaver, J. M. R.

    2003-12-01

    A scheme for the fabrication of combined Scanning Electrochemical Microscopy — Atomic Force Microscopy (SECM-AFM) probes is presented for both silicon nitride and silicon cantilevers. The advantages over exsisting methods used for their production is explained. The process flow is described and initial results from electrodeposition of silver are presented.

  15. Isolation and morphological characterization of ovine amniotic fluid mesenchymal stem cells

    PubMed Central

    Tian, Yunyun; Tao, Li; Zhao, Siriguleng; Tai, Dapeng; Liu, Dongjun; Liu, Pengxia

    2015-01-01

    Mesenchymal stem cells (MSCs) are one of the most promising cell populations for tissue engineering and regenerative medicine. Of utmost importance to MSC research is identification of MSC sources that are easily obtainable and stable. Several studies have shown that MSCs can be isolated from amniotic fluid. The sheep is one of the main types of farm animal, and it has many biophysical and biochemical similarities to humans. Here, we obtained MSCs from ovine amniotic fluid and determined the expansion capacity, surface and intracellular marker expression, karyotype, and multilineage differentiation ability of these ovine amniotic fluid mesenchymal stem cells (oAF-MSCs). Moreover, expression levels of differentiation markers were measured using reverse transcription-qPCR (RT-qPCR). Our phenotypic analysis shows that the isolated oAF-MSCs are indeed MSCs. PMID:26616638

  16. Relationship between automated total nucleated cell count and enumeration of cells on direct smears of canine synovial fluid.

    PubMed

    Dusick, Allison; Young, Karen M; Muir, Peter

    2014-12-01

    Canine osteoarthritis is a common disorder seen in veterinary clinical practice and causes considerable morbidity in dogs as they age. Synovial fluid analysis is an important tool for diagnosis and treatment of canine joint disease and obtaining a total nucleated cell count (TNCC) is particularly important. However, the low sample volumes obtained during arthrocentesis are often insufficient for performing an automated TNCC, thereby limiting diagnostic interpretation. The aim of the present study was to investigate whether estimation of TNCC in canine synovial fluid could be achieved by performing manual cell counts on direct smears of fluid. Fifty-eight synovial fluid samples, taken by arthrocentesis from 48 dogs, were included in the study. Direct smears of synovial fluid were prepared, and hyaluronidase added before cell counts were obtained using a commercial laser-based instrument. A protocol was established to count nucleated cells in a specific region of the smear, using a serpentine counting pattern; the mean number of nucleated cells per 400 × field was then calculated. There was a positive correlation between the automated TNCC and mean manual cell count, with more variability at higher TNCC. Regression analysis was performed to estimate TNCC from manual counts. By this method, 78% of the samples were correctly predicted to fall into one of three categories (within the reference interval, mildly to moderately increased, or markedly increased) relative to the automated TNCC. Intra-observer and inter-observer agreement was good to excellent. The results of the study suggest that interpretation of canine synovial fluid samples of low volume can be aided by methodical manual counting of cells on direct smears. PMID:25439439

  17. Mounting Pressure in the Microenvironment: Fluids, Solids, and Cells in Pancreatic Ductal Adenocarcinoma.

    PubMed

    DuFort, Christopher C; DelGiorno, Kathleen E; Hingorani, Sunil R

    2016-06-01

    The microenvironment influences the pathogenesis of solid tumors and plays an outsized role in some. Our understanding of the stromal response to cancers, particularly pancreatic ductal adenocarcinoma, has evolved from that of host defense to tumor offense. We know that most, although not all, of the factors and processes in the microenvironment support tumor epithelial cells. This reappraisal of the roles of stromal elements has also revealed potential vulnerabilities and therapeutic opportunities to exploit. The high concentration in the stroma of the glycosaminoglycan hyaluronan, together with the large gel-fluid phase and pressures it generates, were recently identified as primary sources of treatment resistance in pancreas cancer. Whereas the relatively minor role of free interstitial fluid in the fluid mechanics and perfusion of tumors has been long appreciated, the less mobile, gel-fluid phase has been largely ignored for historical and technical reasons. The inability of classic methods of fluid pressure measurement to capture the gel-fluid phase, together with a dependence on xenograft and allograft systems that inaccurately model tumor vascular biology, has led to an undue emphasis on the role of free fluid in impeding perfusion and drug delivery and an almost complete oversight of the predominant role of the gel-fluid phase. We propose that a hyaluronan-rich, relatively immobile gel-fluid phase induces vascular collapse and hypoperfusion as a primary mechanism of treatment resistance in pancreas cancers. Similar properties may be operant in other solid tumors as well, so revisiting and characterizing fluid mechanics with modern techniques in other autochthonous cancers may be warranted. PMID:27072672

  18. Fluid intake and incidence of renal cell carcinoma in UK women

    PubMed Central

    Allen, N E; Balkwill, A; Beral, V; Green, J; Reeves, G

    2011-01-01

    Background: It has been suggested that the apparent protective effect of alcohol intake on renal cell carcinoma may be due to the diluting effect of carcinogens by a high total fluid intake. We assessed the association between intakes of total fluids and of specific beverages on the risk of renal cell carcinoma in a large prospective cohort of UK women. Methods: Information on beverage consumption was obtained from a questionnaire sent ∼3 years after recruitment into the Million Women Study. Cox proportional hazards models were used to estimate relative risks (RRs) and 95% confidence intervals (CIs) for renal cell carcinoma associated with beverage consumption adjusted for age, region of residence, socioeconomic status, smoking, and body mass index. Results: After an average of 5.2 years of follow-up, 588 cases of renal cell carcinoma were identified among 779 369 women. While alcohol intake was associated with a reduced risk of renal cell carcinoma (RR for ⩾2 vs <1 drink per day: 0.76; 95% CI: 0.61–0.96; P for trend=0.02), there was no association with total fluid intake (RR for ⩾12 vs <7 drinks per day: 1.15; 95% CI: 0.91–1.45; P for trend=0.3) or with intakes of specific beverages. Conclusions: The apparent protective effect of alcohol on the risk of renal cell carcinoma is unlikely to be related to a high fluid intake. PMID:21407222

  19. Effect of addition of human follicular fluid on progesterone secretion by cultured sheep granulosa cells.

    PubMed

    Kumari, G L; Vohra, S; Raghavan, V

    1982-10-01

    The effect of addition of human follicular fluid to cultures of granulosa cells of large sheep follicles (4-6 mm in diameter) on basal and LH-stimulated progesterone secretion was investigated. Both luteinization and progesterone secretion were inhibited by addition of 10% (w/v) charcoal-treated follicular fluid from medium (2-6 mm) and large (7-16 mm) follicles which had low concentrations of estradiol-17 beta, progesterone and LH. In comparison, the fluid from large follicles, having high levels of the same hormones, stimulated both the parameters, and addition of LH along with the fluid had no further effect. Fluid collected from cystic follicles appeared to be stimulatory which also had elevated levels of estradiol-17 beta and progesterone. These findings indicate the presence of both the inhibitors and stimulators of luteinization in human follicular fluid. The effectiveness of any of them either to inhibit or stimulate luteinization probably will depend upon the composition of the follicular fluid and the stage of maturation of the follicles from which it was collected. PMID:6218983

  20. Cell-free microRNAs in blood and other body fluids, as cancer biomarkers.

    PubMed

    Ortiz-Quintero, Blanca

    2016-06-01

    The discovery of cell-free microRNAs (miRNAs) in serum, plasma and other body fluids has yielded an invaluable potential source of non-invasive biomarkers for cancer and other non-malignant diseases. miRNAs in the blood and other body fluids are highly stable in biological samples and are resistant to environmental conditions, such as freezing, thawing or enzymatic degradation, which makes them convenient as potential biomarkers. In addition, they are more easily sampled than tissue miRNAs. Altered levels of cell-free miRNAs have been found in every type of cancer analysed, and increasing evidence indicates that they may participate in carcinogenesis by acting as cell-to-cell signalling molecules. This review summarizes the biological characteristics and mechanisms of release of cell-free miRNAs that make them promising candidates as non-invasive biomarkers of cancer. PMID:27218664

  1. Electrical conductivity measurements of aqueous fluids under pressure with a hydrothermal diamond anvil cell

    NASA Astrophysics Data System (ADS)

    Ni, Huaiwei; Chen, Qi; Keppler, Hans

    2014-11-01

    Electrical conductivity data of aqueous fluids under pressure can be used to derive the dissociation constants of electrolytes, to assess the effect of ionic dissociation on mineral solubility, and to interpret magnetotelluric data of earth's interior where a free fluid phase is present. Due to limitation on the tensile strength of the alloy material of hydrothermal autoclaves, previous measurements of fluid conductivity were mostly restricted to less than 0.4 GPa and 800 °C. By adapting a Bassett-type hydrothermal diamond anvil cell, we have developed a new method for acquiring electrical conductivity of aqueous fluids under pressure. Our preliminary results for KCl solutions using the new method are consistent with literature data acquired with the conventional method, but the new method has great potential for working in a much broader pressure range.

  2. Spinal fluid lymphocytes responsive to autologous and allogeneic cells in multiple sclerosis and control individuals.

    PubMed Central

    Birnbaum, G; Kotilinek, L; Schwartz, M; Sternad, M

    1984-01-01

    Spinal fluid lymphocytes from multiple sclerosis (MS) patients and controls were stimulated with either autologous non-T cells or with allogeneic non-T cells followed by stimulation with autologous non-T lymphocytes. Cells responding to these stimuli were cloned and their proliferative responses to autologous and allogeneic MS and normal non-T cells were measured. Large numbers of clones with specific patterns of reaction to both autologous and allogeneic cells were obtained from lymphocytes in MS cerebrospinal fluid (CSF), but only occasionally from cells in control CSF. Patterns of responses among clones from a particular CSF were similar and often identical, which suggested that cells in MS CSF were relatively restricted in their specificities. Surface antigen phenotyping of the clones showed them to be predominantly OKT4+, with 13% OKT8+ and 11% OKT4+8+. Peripheral T cells that were stimulated and cultured in parallel with CSF cells were different in that they usually did not give rise to as many clones nor were their patterns of response similar. Many CSF clones were heteroclitic, that is they responded to particular allogeneic cells but not autologous cells. Lymphocytes in MS CSF thus appear to represent a selected population of cells with a high frequency of responsiveness to autologous and allogeneic antigens. Such responses may be evidence for immune regulation within the central nervous system or could represent responses to altered-self antigens. PMID:6237121

  3. Human amniotic fluid derived mesenchymal stem cells cause an anti-cancer effect on breast cancer cell line in vitro.

    PubMed

    Ghafarzadeh, M; Eatemadi, A; Fakhravar, Z

    2016-01-01

    Human amniotic fluid stem cells (hAFSCs) have the ability to self-renew, and multipotent differentiation into three germ layer cells. We obtained 5 ml amniotic fluid from ten 16-20 week pregnant women undergoing amniocentesis. hAFSCs were isolated from all samples, co-cultured with T47D breast cancer cell line and characterized using flow cytometry and RT-PCR. After 3, 4 and 5 days, T47D and HSFCs viability were evaluated with MTT assay. After 5 days of co-culture T47D cells viability were decreased. Our findings showed that hAFSCs can release soluble factors in cell culture, causing an efficient anticancer effect. PMID:27262812

  4. Comparison of equine bone marrow-, umbilical cord matrix and amniotic fluid-derived progenitor cells.

    PubMed

    Lovati, Arianna Barbara; Corradetti, Bruna; Lange Consiglio, Anna; Recordati, Camilla; Bonacina, Elisa; Bizzaro, Davide; Cremonesi, Fausto

    2011-02-01

    The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs) and umbilical cord matrix (EUC-MSCs). It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Bone marrow, umbilical cord and amniotic fluid samples were harvested from different horses. Cells were analyzed for proliferation, immunocytochemical, stem cell gene expression and multilineage plasticity. BM- and AF-MSCs took similar time to reach confluence and showed comparable plating efficiency. All cell lines expressed identical stem cell markers and capability to differentiate towards osteogenic lineage. Almost all cell lines differentiated into the adipogenic lineage as demonstrated by cytochemical staining, even if no adipose gene expression was detectable for AF-MSCs. AF- and EUC-MSCs showed a limited chondrogenic differentiation compared with BM-MSCs as demonstrated by histological and biochemical analyses. These findings suggest that AF-MSCs appeared to be a readily obtainable and highly proliferative cell line from an uninvasive source that may represent a good model system for stem cell biology. More studies are needed to investigate their multilineage potential. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation. PMID:21193959

  5. Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases

    PubMed Central

    Antonucci, Ivana; Provenzano, Martina; Rodrigues, Melissa; Pantalone, Andrea; Salini, Vincenzo; Ballerini, Patrizia; Borlongan, Cesar V.; Stuppia, Liborio

    2016-01-01

    In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers “in vitro”. In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases. PMID:27110774

  6. Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases.

    PubMed

    Antonucci, Ivana; Provenzano, Martina; Rodrigues, Melissa; Pantalone, Andrea; Salini, Vincenzo; Ballerini, Patrizia; Borlongan, Cesar V; Stuppia, Liborio

    2016-01-01

    In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers "in vitro". In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases. PMID:27110774

  7. Detection of Pathogens Using AFM and SPR

    NASA Astrophysics Data System (ADS)

    Vaseashta, Ashok

    2005-03-01

    A priori detection of pathogens in food and water has become a subject of paramount importance. Several recent incidents have resulted in the government passing stringent regulations for tolerable amounts of contamination of food products. Identification and/or monitoring of bacterial contamination in food are critical. The conventional methods of pathogen detection require time-consuming steps to arrive disembark at meaningful measurement in a timely manner as the detection time exceeds the time in which perishable food recycles through the food chain distribution. The aim of this presentation is to outline surface plasmon resonance (SPR) and atomic force microscopy (AFM) as two methods for fast detect6ion of pathogens. Theoretical basis of SPR and experimental results of SPR and AFM on E. coli O157:H7 and prion are presented.

  8. Multiphase modelling of the effect of fluid shear stress on cell yield and distribution in a hollow fibre membrane bioreactor.

    PubMed

    Pearson, Natalie C; Waters, Sarah L; Oliver, James M; Shipley, Rebecca J

    2015-04-01

    We present a simplified two-dimensional model of fluid flow, nutrient transport and cell distribution in a hollow fibre membrane bioreactor, with the aim of exploring how fluid flow can be used to control the distribution and yield of a cell population which is sensitive to both fluid shear stress and nutrient concentration. The cells are seeded in a scaffold in a layer on top of the hollow fibre, only partially occupying the extracapillary space. Above this layer is a region of free-flowing fluid which we refer to as the upper fluid layer. The flow in the lumen and upper fluid layer is described by the Stokes equations, whilst the flow in the porous fibre membrane is assumed to follow Darcy's law. Porous mixture theory is used to model the dynamics of and interactions between the cells, scaffold and fluid in the cell-scaffold construct. The concentration of a limiting nutrient (e.g. oxygen) is governed by an advection-reaction-diffusion equation in each region. Through exploitation of the small aspect ratio of each region and asymptotic analysis, we derive a coupled system of partial differential equations for the cell volume fraction and nutrient concentration. We use this model to investigate the effect of mechanotransduction on the distribution and yield of the cell population, by considering cases in which cell proliferation is either enhanced or limited by fluid shear stress and by varying experimentally controllable parameters such as flow rate and cell-scaffold construct thickness. PMID:25212097

  9. Computational and Experimental Models of Cancer Cell Response to Fluid Shear Stress

    PubMed Central

    Mitchell, Michael J.; King, Michael R.

    2013-01-01

    It has become evident that mechanical forces play a key role in cancer metastasis, a complex series of steps that is responsible for the majority of cancer-related deaths. One such force is fluid shear stress, exerted on circulating tumor cells by blood flow in the vascular microenvironment, and also on tumor cells exposed to slow interstitial flows in the tumor microenvironment. Computational and experimental models have the potential to elucidate metastatic behavior of cells exposed to such forces. Here, we review the fluid-generated forces that tumor cells are exposed to in the vascular and tumor microenvironments, and discuss recent computational and experimental models that have revealed mechanotransduction phenomena that may play a role in the metastatic process. PMID:23467856

  10. Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

    PubMed

    Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

    2012-08-01

    As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy. PMID:22653384

  11. Subjective food hypersensitivity: assessment of enterochromaffin cell markers in blood and gut lavage fluid

    PubMed Central

    Gregersen, Kine; Valeur, Jørgen; Lillestøl, Kristine; Frøyland, Livar; Araujo, Pedro; Lied, Gülen Arslan; Berstad, Arnold

    2011-01-01

    Background: Food hypersensitivity is commonly suspected, but seldom verified. Patients with subjective food hypersensitivity suffer from both intestinal and extraintestinal health complaints. Abnormalities of the enterochromaffin cells may play a role in the pathogenesis. The aim of this study was to investigate enterochromaffin cell function in patients with subjective food hypersensitivity by measuring serum chromogranin A (CgA) and 5-hydroxytryptamine (5-HT, serotonin) in gut lavage fluid. Methods: Sixty-nine patients with subjective food hypersensitivity were examined. Twenty-three patients with inflammatory bowel disease and 35 healthy volunteers were included as comparison groups. CgA was measured in serum by enzyme-linked immunosorbent assay. Gut lavage fluid was obtained by administering 2 L of polyethylene glycol solution intraduodenally. The first clear fluid passed per rectum was collected and 5-HT was analyzed by liquid chromatography tandem mass spectrometry. Results: Serum levels of CgA were significantly lower in patients with subjective food hypersensitivity than in healthy controls (P = 0.04). No differences were found in 5-HT levels in gut lavage fluid between patients with subjective food hypersensitivity and the control groups. There was no correlation between serum CgA and gut lavage 5-HT. Conclusion: Decreased blood levels of CgA suggest neuroendocrine alterations in patients with subjective food hypersensitivity. However, 5-HT levels in gut lavage fluid were normal. PMID:21887108

  12. Phenotypic characteristics of joint fluid cells from patients with continuous joint effusion after total knee arthroplasty.

    PubMed

    Niki, Yasuo; Matsumoto, Hideo; Otani, Toshiro; Yatabe, Taku; Funayama, Atsushi; Maeno, Shinichi; Tomatsu, Taisuke; Toyama, Yoshiaki

    2006-03-01

    Joint effusion after total joint arthroplasty (TJA) is a manifestation of inflammatory reactions within the prosthetic joint. Among the various causes for joint effusion following TJA, deep infection (DI), wear particle-induced synovitis (PS) and metal sensitivity to the implant should be excluded as soon as possible, as these may result in the failure of TJA. The present study analyzed joint fluid cells from patients after total knee arthroplasty (TKA) using fluorescence-activated cell sorter (FACS), and examined the feasibility of using FACS to exclude the possibility of biomaterial-related complication. A total of 72TKAs from 64 patients suffering from joint effusion were examined in this study. Joint fluid was aspirated in outpatient clinics and applied to FACS. The results indicated that patients could be clearly classified into four types based on forward/side scatter profiles. Analysis of specific CD markers revealed that leukocytes were selectively recruited from blood to inflamed prosthetic joints. Dominant cell types were CD16+neutrophils in DI and increased rheumatoid activity, CD14+macrophages in PS, and CD3+CD45RO+T cells in metal sensitivity. These findings suggest the feasibility of diagnosing joint effusion by analyzing dominant cell type recruited using FACS. In conclusion, FACS may offer a useful tool for analyzing joint fluid cells from post-TJA patients and for excluding biomaterial-related complication following TJA. PMID:16183112

  13. Mapping the osteocytic cell response to fluid flow using RNA-Seq.

    PubMed

    Govey, Peter M; Kawasawa, Yuka Imamura; Donahue, Henry J

    2015-12-16

    Bone adaptation to mechanical loading is regulated via signal transduction by mechano-sensing osteocytes. Mineral-embedded osteocytes experience strain-induced interstitial fluid flow and fluid shear stress, and broad shifts in gene expression are key components in the signaling pathways that regulate bone turnover. RNA sequencing analysis, or RNA-Seq, enables more complete characterization of mechano-responsive transcriptome regulation than previously possible. We hypothesized that RNA-Seq of osteocytic MLO-Y4 cells reveals both expected and novel gene transcript regulation in cells previously fluid flowed and analyzed using gene microarrays. MLO-Y4 cells were flowed for 2h with 1Pa oscillating fluid shear stress and post-incubated 2h. RNA-Seq of original samples detected 55 fluid flow-regulated gene transcripts (p-corrected <0.05), the same number previously detected by microarray. However, RNA-Seq demonstrated greater dynamic range, with all 55 transcripts increased >1.5-fold or decreased <0.67-fold whereas 10 of 55 met this cut-off by microarray. Analyses were complimentary in patterns of regulation, though only 6 transcripts were significant in both RNA-Seq and microarray analyses: Cxcl5, Cxcl1, Zc3h12a, Ereg, Slc2a1, and Egln1. As part of a broad inflammatory response inferred by gene ontology analyses, we again observed greatest up-regulation of inflammatory C-X-C motif chemokines, and newly implicated HIF-1α and AMPK signaling pathways. Importantly, we detected both expected fluid flow-sensitive transcripts (e.g. Nos2 [iNOS], Ptgs2 [COX-2], Ccl7) and transcripts not previously identified as flow-sensitive, e.g. Ccl2. We found RNA-Seq advantageous over microarrays because of its greater dynamic range and ability to analyze unbiased estimation of gene expression, informing our understanding of osteocyte signaling. PMID:26573903

  14. Fluid Flow through a High Cell Density Fluidized-Bed during Centrifugal Bioreactor Culture

    PubMed Central

    Detzel, Christopher J.; Van Wie, Bernard J.; Ivory, Cornelius F.

    2010-01-01

    An increasing demand for products such as tissues, proteins, and antibodies from mammalian cell suspension cultures is driving interest in increasing production through high-cell density bioreactors. The centrifugal bioreactor (CCBR) retains cells by balancing settling forces with surface drag forces due to medium throughput and is capable of maintaining cell densities above 108 cells/mL. This article builds on a previous study where the fluid mechanics of an empty CCBR were investigated showing fluid flow is nonuniform and dominated by Coriolis forces, raising concerns about nutrient and cell distribution. In this article, we demonstrate that the previously reported Coriolis forces are still present in the CCBR, but masked by the presence of cells. Experimental dye injection observations during culture of 15 μm hybridoma cells show a continual uniform darkening of the cell bed, indicating the region of the reactor containing cells is well mixed. Simulation results also indicate the cell bed is well mixed during culture of mammalian cells ranging in size from 10 to 20 μm. However, simulations also allow for a slight concentration gradient to be identified and attributed to Coriolis forces. Experimental results show cell density increases from 0.16 to 0.26 when centrifugal force is doubled by increasing RPM from 650 to 920 at a constant inlet velocity of 6.5 cm/s; an effect also observed in the simulation. Results presented in this article indicate cells maintained in the CCBR behave as a high-density fluidized bed of cells providing a homogeneous environment to ensure optimal growth conditions. PMID:20205172

  15. Infectious hematopoietic necrosis virus detected by separation and incubation of cells from salmonid cavity fluid.

    USGS Publications Warehouse

    Mulcahy, D.; Batts, W.N.

    1987-01-01

    Infectious hematopoietic necrosis (IHN) virus is usually detected by inoculating susceptible cell cultures with cavity ("ovarian") fluid (CF) from spawning females. We identified additional adult carriers of virus in spawning populations of steelhead trout (Salmo gairdneri) and sockeye salmon (Oncorhynchus nerka) by collecting nonerythrocytic cells from CF samples by low-speed centrifugation, culturing the cells for at least 7 d at 15 °C, and then testing the culture medium for virus. Virus appeared in the cultured cells from some samples of CF that remained negative during incubation. In additional samples of CF from these species, the virus titer increased in cultured cells compared with the titer in the original CF sample. With chinook salmon (O.tshawytscha), no negative samples converted to positive during incubation, but the virus titer was retained in incubated CF cells, but not in cell-free CF.

  16. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

    PubMed Central

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-01-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies. PMID:27294211

  17. Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds

    PubMed Central

    Skardal, Aleksander; Mack, David; Kapetanovic, Edi; Atala, Anthony; Jackson, John D.; Yoo, James

    2012-01-01

    Stem cells obtained from amniotic fluid show high proliferative capacity in culture and multilineage differentiation potential. Because of the lack of significant immunogenicity and the ability of the amniotic fluid-derived stem (AFS) cells to modulate the inflammatory response, we investigated whether they could augment wound healing in a mouse model of skin regeneration. We used bioprinting technology to treat full-thickness skin wounds in nu/nu mice. AFS cells and bone marrow-derived mesenchymal stem cells (MSCs) were resuspended in fibrin-collagen gel and “printed” over the wound site. At days 0, 7, and 14, AFS cell- and MSC-driven wound closure and re-epithelialization were significantly greater than closure and re-epithelialization in wounds treated by fibrin-collagen gel only. Histological examination showed increased microvessel density and capillary diameters in the AFS cell-treated wounds compared with the MSC-treated wounds, whereas the skin treated only with gel showed the lowest amount of microvessels. However, tracking of fluorescently labeled AFS cells and MSCs revealed that the cells remained transiently and did not permanently integrate in the tissue. These observations suggest that the increased wound closure rates and angiogenesis may be due to delivery of secreted trophic factors, rather than direct cell-cell interactions. Accordingly, we performed proteomic analysis, which showed that AFS cells secreted a number of growth factors at concentrations higher than those of MSCs. In parallel, we showed that AFS cell-conditioned media induced endothelial cell migration in vitro. Taken together, our results indicate that bioprinting AFS cells could be an effective treatment for large-scale wounds and burns. PMID:23197691

  18. Osmotic induction of fluid-phase endocytosis in onion epidermal cells.

    PubMed

    Oparka, K J; Prior, D A; Harris, N

    1990-03-01

    A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplams surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells. PMID:24202101

  19. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  20. Functional Human Podocytes Generated in Organoids from Amniotic Fluid Stem Cells.

    PubMed

    Xinaris, Christodoulos; Benedetti, Valentina; Novelli, Rubina; Abbate, Mauro; Rizzo, Paola; Conti, Sara; Tomasoni, Susanna; Corna, Daniela; Pozzobon, Michela; Cavallotti, Daniela; Yokoo, Takashi; Morigi, Marina; Benigni, Ariela; Remuzzi, Giuseppe

    2016-05-01

    Generating kidney organoids using human stem cells could offer promising prospects for research and therapeutic purposes. However, no cell-based strategy has generated nephrons displaying an intact three-dimensional epithelial filtering barrier. Here, we generated organoids using murine embryonic kidney cells, and documented that these tissues recapitulated the complex three-dimensional filtering structure of glomerular slits in vivo and accomplished selective glomerular filtration and tubular reabsorption. Exploiting this technology, we mixed human amniotic fluid stem cells with mouse embryonic kidney cells to establish three-dimensional chimeric organoids that engrafted in vivo and grew to form vascularized glomeruli and tubular structures. Human cells contributed to the formation of glomerular structures, differentiated into podocytes with slit diaphragms, and internalized exogenously infused BSA, thus attaining in vivo degrees of specialization and function unprecedented for donor stem cells. In conclusion, human amniotic fluid stem cell chimeric organoids may offer new paths for studying renal development and human podocyte disease, and for facilitating drug discovery and translational research. PMID:26516208

  1. The interaction between a solid body and viscous fluid by marker-and-cell method

    NASA Technical Reports Server (NTRS)

    Cheng, R. Y. K.

    1976-01-01

    A computational method for solving nonlinear problems relating to impact and penetration of a rigid body into a fluid type medium is presented. The numerical techniques, based on the Marker-and-Cell method, gives the pressure and velocity of the flow field. An important feature in this method is that the force and displacement of the rigid body interacting with the fluid during the impact and sinking phases are evaluated from the boundary stresses imposed by the fluid on the rigid body. A sample problem of low velocity penetration of a rigid block into still water is solved by this method. The computed time histories of the acceleration, pressure, and displacement of the block show food agreement with experimental measurements. A sample problem of high velocity impact of a rigid block into soft clay is also presented.

  2. Experiment 7: The Geophysical Fluid Flow Cell Experiment on USML-2

    NASA Technical Reports Server (NTRS)

    Hart, John E.; Ohlsen, Daniel R.; Kittleman, Scott; Leslie, Fred W.; Miller, Timothy L.

    1998-01-01

    The Geophysical Fluid Flow Cell (GFFC) experiment performed visualizations of thermal convection in a rotating differentially heated spherical shell of fluid. In these experiments dielectric polarization forces are used to generate a radially directed buoyancy force. This enables the laboratory simulation of a number of geophysically and astrophysically important situations in which sphericity and rotation both impose strong constraints on global scale fluid motions. During USML-2 a large set of experiments with spherically symmetric heating were carried out. These enabled the determination of critical points for the transition to various forms of non-axisymmetric convection and, for highly turbulent flows, the transition latitudes separating the different modes of motion. This paper presents a first analysis of these experiments as well as data on the general performance of the instrument during the USML-2 flight.

  3. Using a co-culture microsystem for cell migration under fluid shear stress.

    PubMed

    Yeh, Chia-Hsien; Tsai, Shen-Hsing; Wu, Li-Wha; Lin, Yu-Cheng

    2011-08-01

    We have successfully developed a microsystem to co-cultivate two types of cells with a minimum defined gap of 50 μm, and to quantitatively study the impact of fluid shear stress on the mutual influence of cell migration velocity and distance. We used the hydrostatic pressure to seed two different cells, endothelial cells (ECs) and smooth muscle cells (SMCs), on opposite sides of various gap sizes (500 μm, 200 μm, 100 μm, and 50 μm). After cultivating the cells for 12 h and peeling the co-culture microchip from the culture dish, we studied the impacts of gap size on the migration of either cell type in the absence or presence of fluid shear stress (7 dyne cm(-2) and 12 dyne cm(-2)) influence. We found that both gap size and shear stress have profound influence on cell migration. Smaller gap sizes (100 μm and 50 μm) significantly enhanced cell migration, suggesting a requirement of an effective concentration of released factor(s) by either cell type in the gap region. Flow-induced shear stress delayed the migration onset of either cell type in a dose-dependent manner regardless of the gap size. Moreover, shear stress-induced decrease of cell migration becomes evident when the gap size was 500 μm. We have developed a co-culture microsystem for two kinds of cells and overcome the conventional difficulties in observation and mixed culture, and it would have more application for bio-manipulation and tissue repair engineering. PMID:21695290

  4. Bone cell mechanosensation of fluid flow stimulation: a fluid-structure interaction model characterising the role integrin attachments and primary cilia.

    PubMed

    Vaughan, T J; Mullen, C A; Verbruggen, S W; McNamara, L M

    2015-08-01

    Load-induced fluid flow acts as an important biophysical signal for bone cell mechanotransduction in vivo, where the mechanical environment is thought to be monitored by integrin and primary cilia mechanoreceptors on the cell body. However, precisely how integrin- and primary cilia-based mechanosensors interact with the surrounding fluid flow stimulus and ultimately contribute to the biochemical response of bone cells within either the in vitro or in vivo environment remains poorly understood. In this study, we developed fluid-structure interaction models to characterise the deformation of integrin- and primary cilia-based mechanosensors in bone cells under fluid flow stimulation. Under in vitro fluid flow stimulation, these models predicted that integrin attachments on the cell-substrate interface were highly stimulated ε(eq) > 200,000 με, while the presence of a primary cilium on the cell also resulted in significant strain amplifications, arising at the ciliary base. As such, these mechanosensors likely play a role in mediating bone mechanotransduction in vitro. Under in vivo fluid flow stimulation, integrin attachments along the canalicular wall were highly stimulated and likely play a role in mediating cellular responses in vivo. The role of the primary cilium as a flow sensor in vivo depended upon its configuration within the lacunar cavity. Specifically, our results showed that a short free-standing primary cilium could not effectively fulfil a flow sensing role in vivo. However, a primary cilium that discretely attaches the lacunar wall can be highly stimulated, due to hydrodynamic pressure in the lacunocanalicular system and, as such, could play a role in mediating bone mechanotransduction in vivo. PMID:25399300

  5. Simulation of Cell Seeding Within a Three-Dimensional Porous Scaffold: A Fluid-Particle Analysis

    PubMed Central

    Olivares, Andy L.

    2012-01-01

    Cell seeding is a critical step in tissue engineering. A high number of cells evenly distributed in scaffolds after seeding are associated with a more functional tissue culture. Furthermore, high cell densities have shown the possibility to reduce culture time or increase the formation of tissue. Experimentally, it is difficult to predict the cell-seeding process. In this study, a new methodology to simulate the cell-seeding process under perfusion conditions is proposed. The cells are treated as spherical particles dragged by the fluid media, where the physical parameters are computed through a Lagrangian formulation. The methodology proposed enables to define the kinetics of cell seeding continuously over time. An exponential relationship was found to optimize the seeding time and the number of cells seeded in the scaffold. The cell distribution and cell efficiency predicted using this methodology were similar to the experimental results of Melchels et al. One of the main advantages of this method is to be able to determine the three-dimensional position of all the seeded cells and to, therefore, better know the initial conditions for further cell proliferation and differentiation studies. This study opens up the field of numerical predictions related to the interactions between biomaterials, cells, and dynamics media. PMID:22372887

  6. Therapeutic outcomes of transplantation of amniotic fluid-derived stem cells in experimental ischemic stroke

    PubMed Central

    Tajiri, Naoki; Acosta, Sandra; Portillo-Gonzales, Gabriel S.; Aguirre, Daniela; Reyes, Stephanny; Lozano, Diego; Pabon, Mibel; Dela Peña, Ike; Ji, Xunming; Yasuhara, Takao; Date, Isao; Solomita, Marianna A.; Antonucci, Ivana; Stuppia, Liborio; Kaneko, Yuji; Borlongan, Cesar V.

    2014-01-01

    Accumulating preclinical evidence suggests the use of amnion as a source of stem cells for investigations of basic science concepts related to developmental cell biology, but also for stem cells’ therapeutic applications in treating human disorders. We previously reported isolation of viable rat amniotic fluid-derived stem (AFS) cells. Subsequently, we recently reported the therapeutic benefits of intravenous transplantation of AFS cells in a rodent model of ischemic stroke. Parallel lines of investigations have provided safety and efficacy of stem cell therapy for treating stroke and other neurological disorders. This review article highlights the need for investigations of mechanisms underlying AFS cells’ therapeutic benefits and discusses lab-to-clinic translational gating items in an effort to optimize the clinical application of the cell transplantation for stroke. PMID:25165432

  7. Hydrothermal diamond-anvil cell: Application to studies of geologic fluids

    USGS Publications Warehouse

    Chou, I.-Ming

    2003-01-01

    The hydrothermal diamond-anvil cell (HDAC) was designed to simulate the geologic conditions of crustal processes in the presence of water or other fluids. The HDAC has been used to apply external pressure to both synthetic and natural fluid inclusions in quartz to minimize problems caused by stretching or decrepitation of inclusions during microthermometric analysis. When the HDAC is loaded with a fluid sample, it can be considered as a large synthetic fluid inclusion and therefore, can be used to study the PVTX properties as well as phase relations of the sample fluid. Because the HDAC has a wide measurement pressure-temperature range and also allows in-situ optical observations, it has been used to study critical phenomena of various chemical systems, such as the geologically important hydrous silicate melts. It is possible, when the HDAC is combined with synchrotron X-ray sources, to obtain basic information on speciation and structure of metal including rare-earth elements (REE) complexes in hydrothermal solutions as revealed by X-ray absorption fine structure (XAFS) spectra. Recent modifications of the HDAC minimize the loss of intensity of X-rays due to scattering and absorption by the diamonds. These modifications are especially important for studying elements with absorption edges below 10 keV and therefore particularly valuable for our understanding of transport and deposition of first-row transition elements and REE in hydrothermal environments.

  8. An altered repertoire of T cell receptor V gene expression by rheumatoid synovial fluid T lymphocytes.

    PubMed

    Lunardi, C; Marguerie, C; So, A K

    1992-12-01

    The pattern of T cell receptor V gene expression by lymphocytes from rheumatoid synovial fluid and paired peripheral blood samples was compared using a polymerase chain reaction (PCR)-based assay. Eight rheumatoid arthritis (RA) patients who had varying durations of disease (from 2 to 20 years) were studied. In all patients there was evidence of a different pattern of V gene expression between the two compartments. Significantly increased expression of at least one V alpha or V beta gene family by synovial fluid T cells was observed in all the patients studied. Three different V alpha (V alpha 10, 15 and 18) and three V beta (V beta 4, 5 and 13) families were commonly elevated. Sequencing of synovial V beta transcripts demonstrated that the basis of increased expression of selected V gene families in the synovial fluid was due to the presence of dominant clonotypes within those families, which constituted up to 53% of the sequences isolated from one particular synovial V gene family. There were considerable differences in the NDJ sequences found in synovial and peripheral blood T cell receptor (TCR) transcripts of the same V beta gene family. These data suggest that the TCR repertoire in the two compartments differs, and that antigen-driven expansion of particular synovial T cell populations is a component of rheumatoid synovitis, and is present in all stages of the disease. PMID:1458680

  9. Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells.

    PubMed

    Vaidyanathan, Ramanathan; Rauf, Sakandar; Dray, Eloïse; Shiddiky, Muhammad J A; Trau, Matt

    2014-03-24

    We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over nonspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific adsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications. PMID:24677444

  10. BOREAS AFM-6 Surface Meteorological Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from the National Oceanic and Atmospheric Adminsitration/Environment Technology Laboratory (NOAA/ETL) collected surface meteorological data from 21 May to 20 Sep 1994 near the Southern Study Area-Old Jack Pine (SSA-OJP) tower site. The data are in tabular ASCII files. The surface meteorological data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  11. Fluid dynamics and noise in bacterial cell–cell and cell–surface scattering

    PubMed Central

    Drescher, Knut; Dunkel, Jörn; Cisneros, Luis H.; Ganguly, Sujoy; Goldstein, Raymond E.

    2011-01-01

    Bacterial processes ranging from gene expression to motility and biofilm formation are constantly challenged by internal and external noise. While the importance of stochastic fluctuations has been appreciated for chemotaxis, it is currently believed that deterministic long-range fluid dynamical effects govern cell–cell and cell–surface scattering—the elementary events that lead to swarming and collective swimming in active suspensions and to the formation of biofilms. Here, we report direct measurements of the bacterial flow field generated by individual swimming Escherichia coli both far from and near to a solid surface. These experiments allowed us to examine the relative importance of fluid dynamics and rotational diffusion for bacteria. For cell–cell interactions it is shown that thermal and intrinsic stochasticity drown the effects of long-range fluid dynamics, implying that physical interactions between bacteria are determined by steric collisions and near-field lubrication forces. This dominance of short-range forces closely links collective motion in bacterial suspensions to self-organization in driven granular systems, assemblages of biofilaments, and animal flocks. For the scattering of bacteria with surfaces, long-range fluid dynamical interactions are also shown to be negligible before collisions; however, once the bacterium swims along the surface within a few microns after an aligning collision, hydrodynamic effects can contribute to the experimentally observed, long residence times. Because these results are based on purely mechanical properties, they apply to a wide range of microorganisms. PMID:21690349

  12. Contracting bubbles in Hele-Shaw cells with a power-law fluid

    NASA Astrophysics Data System (ADS)

    McCue, Scott W.; King, John R.

    2011-02-01

    The problem of bubble contraction in a Hele-Shaw cell is studied for the case in which the surrounding fluid is of power-law type. A small perturbation of the radially symmetric problem is first considered, focussing on the behaviour just before the bubble vanishes, it being found that for shear-thinning fluids the radially symmetric solution is stable, while for shear-thickening fluids the aspect ratio of the bubble boundary increases. The borderline (Newtonian) case considered previously is neutrally stable, the bubble boundary becoming elliptic in shape with the eccentricity of the ellipse depending on the initial data. Further light is shed on the bubble contraction problem by considering a long thin Hele-Shaw cell: for early times the leading-order behaviour is one-dimensional in this limit; however, as the bubble contracts its evolution is ultimately determined by the solution of a Wiener-Hopf problem, the transition between the long thin limit and the extinction limit in which the bubble vanishes being described by what is in effect a similarity solution of the second kind. This same solution describes the generic (slit-like) extinction behaviour for shear-thickening fluids, the interface profiles that generalize the ellipses that characterize the Newtonian case being constructed by the Wiener-Hopf calculation.

  13. A software tool for STED-AFM correlative super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Koho, Sami; Deguchi, Takahiro; Löhmus, Madis; Näreoja, Tuomas; Hänninen, Pekka E.

    2015-03-01

    Multi-modal correlative microscopy allows combining the strengths of several imaging techniques to provide unique contrast. However it is not always straightforward to setup instruments for such customized experiments, as most microscope manufacturers use their own proprietary software, with limited or no capability to interface with other instruments - this makes correlation of the multi-modal data extremely challenging. We introduce a new software tool for simultaneous use of a STimulated Emission Depletion (STED) microscope with an Atomic Force Microscope (AFM). In our experiments, a Leica TCS STED commercial super-resolution microscope, together with an Agilent 5500ilm AFM microscope was used. With our software, it is possible to synchronize the data acquisition between the STED and AFM instruments, as well as to perform automatic registration of the AFM images with the super-resolution STED images. The software was realized in LabVIEW; the registration part was also implemented as an ImageJ script. The synchronization was realized by controlling simple trigger signals, also available in the commercial STED microscope, with a low-cost National Instruments USB-6501 digital I/O card. The registration was based on detecting the positions of the AFM tip inside the STED fieldof-view, which were then used as registration landmarks. The registration should work on any STED and tip-scanning AFM microscope combination, at nanometer-scale precision. Our STED-AFM correlation method has been tested with a variety of nanoparticle and fixed cell samples. The software will be released under BSD open-source license.

  14. Fluid shear stress induces differentiation of Flk-1-positive embryonic stem cells into vascular endothelial cells in vitro.

    PubMed

    Yamamoto, Kimiko; Sokabe, Takaaki; Watabe, Tetsuro; Miyazono, Kohei; Yamashita, Jun K; Obi, Syotaro; Ohura, Norihiko; Matsushita, Akiko; Kamiya, Akira; Ando, Joji

    2005-04-01

    Pluripotent embryonic stem (ES) cells are capable of differentiating into all cell lineages, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. In this study, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1(+)) mouse ES cells were subjected to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S and G(2)-M phases of the cell cycle than Flk-1(+) ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, vascular endothelial cadherin, and PECAM-1 at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker smooth muscle alpha-actin, blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1(+) ES cells into the endothelial cell lineage. The shear stressed Flk-1(+) ES cells formed tubelike structures in collagen gel and developed an extensive tubular network significantly faster than the static controls. Shear stress induced tyrosine phosphorylation of Flk-1 in Flk-1(+) ES cells that was blocked by a Flk-1 kinase inhibitor, SU1498, but not by a neutralizing antibody against VEGF. SU1498 also abolished the shear stress-induced proliferation and differentiation of Flk-1(+) ES cells, indicating that a ligand-independent activation of Flk-1 plays an important role in the shear stress-mediated proliferation and differentiation by Flk-1(+) ES cells. PMID:15576436

  15. Insights Into Fetal and Neonatal Development Through Analysis of Cell-Free RNA in Body Fluids

    PubMed Central

    Bianchi, Diana W.; Maron, Jill L.; Johnson, Kirby L.

    2010-01-01

    The use of cell-free nucleic acids in the circulation of pregnant women for noninvasive prenatal diagnosis is arguably one of the hottest current topics in prenatal medicine. Between 1997 and the present era this field has gone from basic research to clinical application for diagnosis of fetal gender and Rhesus D status. Over the next few years it is likely that noninvasive prenatal diagnosis for Down syndrome will also be possible. Here we summarize current and future clinical applications of analyzing cell-free fetal DNA and RNA in both maternal and neonatal body fluids, including maternal plasma, serum, whole blood, amniotic fluid, and neonatal saliva. We describe methods to evaluate normal and abnormal fetal and neonatal development using gene expression microarrays. We also discuss the ways in which differentially-regulated gene lists can advance knowledge of both fetal and neonatal biology, as well as suggest novel possibilities for fetal and neonatal treatment. PMID:20851538

  16. Peripheral blood and synovial fluid T cells differ in their response to alloantigens and recall antigens presented by dendritic cells.

    PubMed Central

    Stagg, A J; Harding, B; Hughes, R A; Keat, A; Knight, S C

    1991-01-01

    Properties of T cells from inflammatory lesions were analysed by comparing the response of peripheral blood (PB) and synovial fluid (SF) T cells from 19 patients with a range of arthropathies to enriched allogeneic dendritic cells (DC) in a primary mixed leucocyte reaction (MLR). In 17 patients the proliferative response of SF T cells was significantly (P less than 0.05) less than that of PB lymphocytes. The reduced response of SF T cells was observed in all disease categories studied and could not be attributed to differences in cell number requirements or response kinetics. Addition of recombinant interleukin-2 enhanced the response of SF T cells in a dose-dependent manner. Cell mixing experiments suggested that active suppression was not the underlying mechanism of the poor MLR response of SF T cells. In contrast to the MLR response. SF T cells were able to mount vigorous proliferative responses to recall antigen presented by autologous antigen-presenting cells. The possibility is discussed that T cells compartmentalized at inflammatory lesions are a unique population with a diminished ability to interact with DC and respond to primary stimuli but an ability to respond to secondary antigenic challenge. PMID:1826648

  17. Glutathione and GSH-dependent enzymes in bronchoalveolar lavage fluid cells in response to ozone

    SciTech Connect

    Boehme, D.S.; Hotchkiss, J.A.; Henderson, R.F. )

    1992-02-01

    The purpose of this study was to determine if in vivo ozone exposure results in elevations in the levels of glutathione and glutathione-dependent enzymes in cells derived from bronchoalveolar lavage fluid (BALF). Our hypothesis was that, as part of a defense mechanism against oxygen toxicity, such cells would have increased levels of glutathione (GSH) in response to an oxidant stress. Female F344/N rats were exposed to 0.8 ppm ozone, 6 hr/day, for 1, 3, or 7 days, after which cells were collected by lung lavage. The GSH and GSH-peroxidase activity per milligram of protein in the cellular fraction, both necessary for reducing cellular peroxides, were elevated after 3 days of ozone exposure. After 7 days of exposure, cellular GSH had returned to control values, but the activity of glutathione reductase, the enzyme that reduces oxidized glutathione to GSH, was increased. Extracellular GSH concentration and glutathione reductase activity in BALF were also increased after 7 days of exposure. The total glutathione equivalents (GSH and GSSG, both cellular and extracellular) in BALF increased throughout the 7-day exposure, with GSH increasing first in the cells, and then in the extracellular fluid. This study demonstrated that the glutathione anti-oxidant system of BALF cells is stimulated by exposure to ozone. This response may serve to protect cells from the toxic effects of oxidant stress.

  18. Amniotic Fluid-Derived Stem Cells for Cardiovascular Tissue Engineering Applications

    PubMed Central

    Petsche Connell, Jennifer; Camci-Unal, Gulden; Khademhosseini, Ali

    2013-01-01

    Recent research has demonstrated that a population of stem cells can be isolated from amniotic fluid removed by amniocentesis that are broadly multipotent and nontumorogenic. These amniotic fluid-derived stem cells (AFSC) could potentially provide an autologous cell source for treatment of congenital defects identified during gestation, particularly cardiovascular defects. In this review, the various methods of isolating, sorting, and culturing AFSC are compared, along with techniques for inducing differentiation into cardiac myocytes and endothelial cells. Although research has not demonstrated complete and high-yield cardiac differentiation, AFSC have been shown to effectively differentiate into endothelial cells and can effectively support cardiac tissue. Additionally, several tissue engineering and regenerative therapeutic approaches for the use of these cells in heart patches, injection after myocardial infarction, heart valves, vascularized scaffolds, and blood vessels are summarized. These applications show great promise in the treatment of congenital cardiovascular defects, and further studies of isolation, culture, and differentiation of AFSC will help to develop their use for tissue engineering, regenerative medicine, and cardiovascular therapies. PMID:23350771

  19. [Cultivation of amniotic fluid cells for the purpose of diagnosing certain metabolic diseases].

    PubMed

    Zolotukhina, T V; Tsvetkova, I V

    1980-03-01

    A method for preparing amniotic fluid cell culture has been developed. The optimal conditions for the culture involved the incubation period averaging 17.9 days. Normal activity values were obtained for 8 glycosidases whose deficiency is responsible for the development of hereditary accumulation diseases. The data obtained allow a prenatal diagnosis of glycolipidoses (Tay-Sachs, Fabrys, Gaucher's diseases, etc.). The evidence on prenatal diagnosis of Tay-Sach's disease is presented. PMID:7388145

  20. A cell-based sensor of fluid shear stress for microfluidics.

    PubMed

    Varma, Sarvesh; Voldman, Joel

    2015-03-21

    Microsystems designed for cell-based studies or applications inherently require fluid handling. Flows within such systems inevitably generate fluid shear stress (FSS) that may adversely affect cell health. Simple assays of cell viability, morphology or growth are typically reported to indicate any gross disturbances to cell physiology. However, no straightforward metric exists to specifically evaluate physiological implications of FSS within microfluidic devices, or among competing microfluidic technologies. This paper presents the first genetically encoded cell sensors that fluoresce in a quantitative fashion upon FSS pathway activation. We picked a widely used cell line (NIH3T3s) and created a transcriptional cell-sensor where fluorescence turns on when transcription of a relevant FSS-induced protein is initiated. Specifically, we chose Early Growth Factor-1 (a mechanosensitive protein) upregulation as the node for FSS detection. We verified our sensor pathway specificity and functionality by noting induced fluorescence in response to chemical induction of the FSS pathway, seen both through microscopy and flow cytometry. Importantly, we found our cell sensors to be inducible by a range of FSS intensities and durations, with a limit of detection of 2 dynes cm(-2) when applied for 30 minutes. Additionally, our cell-sensors proved their versatility by showing induction sensitivity when made to flow through an inertial microfluidic device environment with typical flow conditions. We anticipate these cell sensors to have wide application in the microsystems community, allowing the device designer to engineer systems with acceptable FSS, and enabling the end-user to evaluate the impact of FSS upon their assay of interest. PMID:25648195

  1. Ketoconazole activates Cl- conductance and blocks Cl- and fluid absorption by cultured cystic fibrosis (CFPAC-1) cells.

    PubMed Central

    Kersting, U; Kersting, D; Spring, K R

    1993-01-01

    The role of arachidonic acid metabolites in the regulation of apical cell membrane Cl- conductance and transepithelial transport of fluid and Cl- by cultured pancreatic cells from cystic fibrosis (CFPAC-1) and corrected (PAC-1) cell lines was evaluated by the use of inhibitors. CFPAC-1 cells did not exhibit an apical membrane Cl- conductance, absorbed Cl- and fluid, and did not respond to stimulation or inhibition of cAMP action. PAC-1 cells exhibited a cAMP-responsive apical Cl- conductance, which was blocked by indomethacin, a cyclooxygenase inhibitor. Ketoconazole, an epoxygenase inhibitor, had virtually no effects on PAC-1 cell Cl- conductance but caused CFPAC-1 cells to develop a cAMP-insensitive Cl- conductance, blocked Cl- and fluid absorption, and reduced transepithelial electrical resistance. Ketoconazole treatment effectively reversed the cystic fibrosis defect in these cultured cells. PMID:7683418

  2. Differential proteomics analysis of mononuclear cells in cerebrospinal fluid of Parkinson’s disease

    PubMed Central

    Xing, Lifei; Wang, Dongtao; Wang, Lihong; Lan, Wenjie; Pan, Suyue

    2015-01-01

    Parkinson’s disease (PD) is one common neurodegenerative disease featured with degeneration of dopaminergic neurons in substantia nigra. Multiple factors participate in the pathogenesis and progression of PD. In this study, we investigated the proteomics profiles of mononuclear cells in cerebrospinal fluids from both PD patients and normal people, in order to explore the correlation between disease factors and PD. Cerebrospinal fluid samples were collected from both PD and normal people and were separated for mononuclear cells in vitro. Proteins were then extracted and separated by 2-dimensional gel electrophoresis. Proteins with differential expressions were identified by comparison to standard proteome expression profile map, followed by software and database analysis. In PD patients, there were 8 proteins with consistent expression profile and 16 proteins with differential expressions. Those differential proteins identified include cytoskeleton proteins (actin, myosin), signal transduction proteins (adenosine cyclase binding protein 1, calcium binding protein, talin) and anti-oxidation factor (thioredoxin peroxide reductase). PD patients had differential protein expressional profiles in the mononuclear cells of cerebrospinal fluids compared to normal people, suggesting the potential involvement of cytoskeleton and signal transduction proteins in apoptosis of neuronal apoptosis and PD pathogenesis. PMID:26823915

  3. Polymer dynamics and fluid flow in actin-based cell motility

    NASA Astrophysics Data System (ADS)

    Theriot, Julie

    2005-03-01

    In living cells, nonequilibrium protein polymerization reactions are frequently used to convert chemical energy into mechanical energy and thereby generate useful force for cellular movements. We have examined the polymer and fluid dynamics in two biological cases where the assembly of branched actin filament networks generates force: the intracellular movement of the bacterial pathogen Listeria monocytogenes, and the extension of the leading edge of skin epithelial cells during wound-healing. In both cases, net actin filament assembly occurs at the front of the network structure and net disassembly occurs at the rear. Actin protein subunits and other network components must be recycled through the fluid phase to the front of the polymerizing network in order for forward movement to continue at steady state. For actin-based movement of Listeria monocytogenes, we have found that actin recycling is not rate-limiting; instead, the speed of movement is governed by the cooperative dissociation of groups of noncovalent protein-protein bonds attaching the filamentous network to the bacterial surface. In contrast, rapid actin-based extension at the leading edge of moving epithelial cells is associated with unusual perturbations in intracellular fluid flow.

  4. Mechanism of vibration-induced repulsion force on a particle in a viscous fluid cell.

    PubMed

    Saadatmand, Mehrrad; Kawaji, Masahiro

    2013-08-01

    Space platforms such as the Space Shuttle and International Space Station have been considered an ideal environment for production of protein and semiconductor crystals of superior quality due to the negligible gravity-induced convection. Although it was believed that under microgravity environment diffusive mass transport would dominate the growth of the crystals, some related experiments have not shown satisfactory results possibly due to the movement of the growing crystals in fluid cells caused by small vibrations present in the space platforms called g-jitter. In ground-based experiments, there have been clear observations of attraction and repulsion of a solid particle with respect to a nearby wall of the fluid cell due to small vibrations. The present work is a numerical investigation on the physical mechanisms responsible for the repulsion force, which has been predicted to increase with the cell vibration frequency and amplitude, as well as the fluid viscosity. Moreover, the simulations have revealed that the repulsion force occurs mostly due to the increased pressure in the narrow gap between the particle and the nearest wall. PMID:24032936

  5. Mechanism of vibration-induced repulsion force on a particle in a viscous fluid cell

    NASA Astrophysics Data System (ADS)

    Saadatmand, Mehrrad; Kawaji, Masahiro

    2013-08-01

    Space platforms such as the Space Shuttle and International Space Station have been considered an ideal environment for production of protein and semiconductor crystals of superior quality due to the negligible gravity-induced convection. Although it was believed that under microgravity environment diffusive mass transport would dominate the growth of the crystals, some related experiments have not shown satisfactory results possibly due to the movement of the growing crystals in fluid cells caused by small vibrations present in the space platforms called g-jitter. In ground-based experiments, there have been clear observations of attraction and repulsion of a solid particle with respect to a nearby wall of the fluid cell due to small vibrations. The present work is a numerical investigation on the physical mechanisms responsible for the repulsion force, which has been predicted to increase with the cell vibration frequency and amplitude, as well as the fluid viscosity. Moreover, the simulations have revealed that the repulsion force occurs mostly due to the increased pressure in the narrow gap between the particle and the nearest wall.

  6. [AFM-based technologies as the way towards the reverse Avogadro number].

    PubMed

    Pleshakova, T O; Shumov, I D; Ivanov, Yu D; Malsagova, K A; Kaysheva, A L; Archakov, A I

    2015-01-01

    Achievement of the concentration detection limit for proteins at the level of the reverse Avogadro number determines the modern development of proteomics. In this review, the possibility of approximating the reverse Avogadro number by using nanotechnological methods (AFM-based fishing with mechanical and electrical stimulation, nanowire detectors, and other methods) are discussed. The ability of AFM to detect, count, visualize and characterize physico-chemical properties of proteins at concentrations up to 10(-17)-10(-18) M is demonstrated. The combination of AFM-fishing with mass-spectrometry allows the identification of proteins not only in pure solutions, but also in multi-component biological fluids (serum). The possibilities to improve the biospecific fishing efficiency by use of SOMAmers in both AFM and nanowire systems are discussed. The paper also provides criteria for evaluation of the sensitivity of fishing-based detection systems. The fishing efficiency depending on the detection system parameters is estimated. The practical implementation of protein fishing depending on the ratio of the sample solution volume and the surface of the detection system is discussed. The advantages and disadvantages of today's promising nanotechnological protein detection methods implemented on the basis of these schemes. PMID:25978390

  7. Clinical relevance and contemporary methods for counting blood cells in body fluids suspected of inflammatory disease.

    PubMed

    Fleming, Chérina; Russcher, Henk; Lindemans, Jan; de Jonge, Robert

    2015-10-01

    In many inflammatory diseases, the cellular components in body fluids [cerebrospinal fluid (CSF), serous fluids] are increased, rendering essential diagnostic information. The diagnostic value of the total white blood cell count (WBC) and differential count has been evaluated extensively over the years, and a remarkable amount of knowledge has been gained; yet, there is a great deal of clinical uncertainty whether the diagnosis should be based solely on these variables. In some diseases, such as peritonitis, the total WBC and differential count has high sensitivity; whereas, in differentiating pleural effusions, it lacks the sensitivity required to be clinically useful. Nevertheless, many guidelines consider these tests as cornerstone parameters, and in combination with clinical variables, they can successfully guide clinical decision making in initiating or postponing a treatment course for infection and/or inflammatory diseases while awaiting culture results. Although other methods are available for detecting and differentiating WBCs in body fluids, manual microscopy is still considered the gold standard despite its many limitations. During the last decade, automated analyzers have become a popular method for first line screening. Continued progress in their design has led to major improvements including their speed, improved accuracy and lower variability compared with microscopy. Disadvantages of this method include high imprecision in low ranges (depending on the method) and interfering factors. In a time where automation is at the front line in clinical laboratories, it is essential the results obtained are precise, accurate and reproducible. This review provides an overview of the relevance for cell counting in a variety of diagnostic body fluids, and highlights the current technologies used. PMID:25879321

  8. Single cell rheometry with a microfluidic constriction: Quantitative control of friction and fluid leaks between cell and channel walls

    PubMed Central

    Preira, Pascal; Valignat, Marie-Pierre; Bico, José; Théodoly, Olivier

    2013-01-01

    We report how cell rheology measurements can be performed by monitoring the deformation of a cell in a microfluidic constriction, provided that friction and fluid leaks effects between the cell and the walls of the microchannels are correctly taken into account. Indeed, the mismatch between the rounded shapes of cells and the angular cross-section of standard microfluidic channels hampers efficient obstruction of the channel by an incoming cell. Moreover, friction forces between a cell and channels walls have never been characterized. Both effects impede a quantitative determination of forces experienced by cells in a constriction. Our study is based on a new microfluidic device composed of two successive constrictions, combined with optical interference microscopy measurements to characterize the contact zone between the cell and the walls of the channel. A cell squeezed in a first constriction obstructs most of the channel cross-section, which strongly limits leaks around cells. The rheological properties of the cell are subsequently probed during its entry in a second narrower constriction. The pressure force is determined from the pressure drop across the device, the cell velocity, and the width of the gutters formed between the cell and the corners of the channel. The additional friction force, which has never been analyzed for moving and constrained cells before, is found to involve both hydrodynamic lubrication and surface forces. This friction results in the existence of a threshold for moving the cells and leads to a non-linear behavior at low velocity. The friction force can nevertheless be assessed in the linear regime. Finally, an apparent viscosity of single cells can be estimated from a numerical prediction of the viscous dissipation induced by a small step in the channel. A preliminary application of our method yields an apparent loss modulus on the order of 100 Pa s for leukocytes THP-1 cells, in agreement with the literature data. PMID:24404016

  9. Natural killer cell subsets in cerebrospinal fluid of patients with multiple sclerosis.

    PubMed

    Rodríguez-Martín, E; Picón, C; Costa-Frossard, L; Alenda, R; Sainz de la Maza, S; Roldán, E; Espiño, M; Villar, L M; Álvarez-Cermeño, J C

    2015-05-01

    Changes in blood natural killer (NK) cells, important players of the immune innate system, have been described in multiple sclerosis (MS). We studied percentages and total cell counts of different effector and regulatory NK cells in cerebrospinal fluid (CSF) of MS patients and other neurological diseases to gain clearer knowledge of the role of these cells in neuroinflammation. NK cell subsets were assessed by flow cytometry in CSF of 85 consecutive MS patients (33 with active disease and 52 with stable MS), 16 with other inflammatory diseases of the central nervous system (IND) and 17 with non-inflammatory neurological diseases (NIND). MS patients showed a decrease in percentages of different CSF NK subpopulations compared to the NIND group. However, absolute cell counts showed a significant increase of all NK subsets in MS and IND patients, revealing that the decrease in percentages does not reflect a real reduction of these immune cells. Remarkably, MS patients showed a significant increase of regulatory/effector (CD56(bright) /CD56(dim) ) NK ratio compared to IND and NIND groups. In addition, MS activity associated with an expansion of NK T cells. These data show that NK cell subsets do not increase uniformly in all inflammatory neurological disease and suggest strongly that regulatory CD56(bright) and NK T cells may arise in CSF of MS patients as an attempt to counteract the CNS immune activation characteristic of the disease. PMID:25565222

  10. Natural killer cell subsets in cerebrospinal fluid of patients with multiple sclerosis

    PubMed Central

    Rodríguez-Martín, E; Picón, C; Costa-Frossard, L; Alenda, R; Sainz de la Maza, S; Roldán, E; Espiño, M; Villar, L M; Álvarez-Cermeño, J C

    2015-01-01

    Changes in blood natural killer (NK) cells, important players of the immune innate system, have been described in multiple sclerosis (MS). We studied percentages and total cell counts of different effector and regulatory NK cells in cerebrospinal fluid (CSF) of MS patients and other neurological diseases to gain clearer knowledge of the role of these cells in neuroinflammation. NK cell subsets were assessed by flow cytometry in CSF of 85 consecutive MS patients (33 with active disease and 52 with stable MS), 16 with other inflammatory diseases of the central nervous system (IND) and 17 with non-inflammatory neurological diseases (NIND). MS patients showed a decrease in percentages of different CSF NK subpopulations compared to the NIND group. However, absolute cell counts showed a significant increase of all NK subsets in MS and IND patients, revealing that the decrease in percentages does not reflect a real reduction of these immune cells. Remarkably, MS patients showed a significant increase of regulatory/effector (CD56bright/CD56dim) NK ratio compared to IND and NIND groups. In addition, MS activity associated with an expansion of NK T cells. These data show that NK cell subsets do not increase uniformly in all inflammatory neurological disease and suggest strongly that regulatory CD56bright and NK T cells may arise in CSF of MS patients as an attempt to counteract the CNS immune activation characteristic of the disease. PMID:25565222

  11. Multiparametric high-resolution imaging of native proteins by force-distance curve-based AFM.

    PubMed

    Pfreundschuh, Moritz; Martinez-Martin, David; Mulvihill, Estefania; Wegmann, Susanne; Muller, Daniel J

    2014-05-01

    A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in force-distance (FD) curve-based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1-3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution. PMID:24743419

  12. Mechanical interaction between cells and fluid for bone tissue engineering scaffold: modulation of the interfacial shear stress.

    PubMed

    Blecha, L D; Rakotomanana, L; Razafimahery, F; Terrier, A; Pioletti, D P

    2010-03-22

    An analytical model of the fluid/cell mechanical interaction was developed. The interfacial shear stress, due to the coupling between the fluid and the cell deformation, was characterized by a new dimensionless number N(fs). For N(fs) above a critical value, the fluid/cell interaction had a damping effect on the interfacial shear stress. Conversely, for N(fs) below this critical value, interfacial shear stress was amplified. As illustration, the role of the dynamic fluid/cell mechanical coupling was studied in a specific biological situation involving cells seeded in a bone scaffold. For the particular bone scaffold chosen, the dimensionless number N(fs) was higher than the critical value. In this case, the dynamic shear stress at the fluid/cell interface is damped for increasing excitation frequency. Interestingly, this damping effect is correlated to the pore diameter of the scaffold, furnishing thus target values in the design of the scaffold. Correspondingly, an efficient cell stimulation might be achieved with a scaffold of pore size larger than 300 microm as no dynamic damping effect is likely to take place. The analytical model proposed in this study, while being a simplification of a fluid/cell mechanical interaction, brings complementary insights to numerical studies by analyzing the effect of different physical parameters. PMID:20004397

  13. Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response.

    PubMed

    Moorefield, Emily C; McKee, Elizabeth E; Solchaga, Luis; Orlando, Guisseppe; Yoo, James J; Walker, Steve; Furth, Mark E; Bishop, Colin E

    2011-01-01

    Amniotic fluid stem (AFS) cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC) show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR) and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family members as well as interleukin-6 (IL-6). AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α), MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and absence of

  14. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    SciTech Connect

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  15. Peritoneal fluid immunocytochemistry used for the diagnosis of a possible case of equine gastrointestinal B-cell lymphoma.

    PubMed

    Duran, Maria Carolina; Starrak, Gregory; Dickinson, Ryan; Montgomery, Julia

    2016-06-01

    After physical examination, ultrasonographic evaluation of thorax and abdomen, and peritoneal fluid analysis, gastrointestinal neoplasia with suspected diffuse peritoneal metastasis was diagnosed in a 17-year-old Arabian gelding. The owner elected euthanasia and declined postmortem examination. Immunocytochemistry analysis of the peritoneal fluid resulted in a diagnosis of B-cell lymphoma. PMID:27247458

  16. Proteomic analysis of Staphylococcus aureus biofilm cells grown under physiologically relevant fluid shear stress conditions

    PubMed Central

    2014-01-01

    Background The biofilm forming bacterium Staphylococcus aureus is responsible for maladies ranging from severe skin infection to major diseases such as bacteremia, endocarditis and osteomyelitis. A flow displacement system was used to grow S. aureus biofilms in four physiologically relevant fluid shear rates (50, 100, 500 and 1000 s-1) to identify proteins that are associated with biofilm. Results Global protein expressions from the membrane and cytosolic fractions of S. aureus biofilm cells grown under the above shear rate conditions are reported. Sixteen proteins in the membrane-enriched fraction and eight proteins in the cytosolic fraction showed significantly altered expression (p < 0.05) under increasing fluid shear. These 24 proteins were identified using nano-LC-ESI-MS/MS. They were found to be associated with various metabolic functions such as glycolysis / TCA pathways, protein synthesis and stress tolerance. Increased fluid shear stress did not influence the expression of two important surface binding proteins: fibronectin-binding and collagen-binding proteins. Conclusions The reported data suggest that while the general metabolic function of the sessile bacteria is minimal under high fluid shear stress conditions, they seem to retain the binding capacity to initiate new infections. PMID:24855455

  17. Cell profile of BAL fluid in children and adolescents with and without lung disease.

    PubMed

    Picinin, Isabela Furtado de Mendonça; Camargos, Paulo Augusto Moreira; Marguet, Christophe

    2010-01-01

    The objective of this study was to review the literature on bronchoalveolar lavage fluid cell profiles in healthy children and adolescents, as well as on the use of BAL as a diagnostic and follow-up tool for lung disease patients in this age bracket. To that end, we used the Medline database, compiling studies published between 1989 and 2009 employing the following MeSH descriptors (with Boolean operators) as search terms: bronchoalveolar lavage AND cytology OR cell AND child. In healthy children, the cell profile includes alveolar macrophages (> 80%), lymphocytes (approximately 10%), neutrophils (approximately 2%) and eosinophils (< 1%). The profile varies depending on the disease under study. The number of neutrophils is greater in wheezing children, especially in non-atopic children, as well as in those with pulmonary infectious and inflammatory profiles, including cystic fibrosis and interstitial lung disease. Eosinophil counts are elevated in children/adolescents with asthma and can reach high levels in those with allergic bronchopulmonary aspergillosis or eosinophilic syndromes. In a heterogeneous group of diseases, the number of lymphocytes can increase. Evaluation of the BAL fluid cell profile, when used in conjunction with clinical and imaging findings, has proven to be an essential tool in the investigation of various lung diseases. Less invasive than transbronchial and open lung biopsies, BAL has great clinical value. Further studies adopting standard international protocols should be carried out. Such studies should involve various age groups and settings in order to obtain reference values for BAL fluid cell profiles, which are necessary for a more accurate interpretation of findings in children and adolescents with lung diseases. PMID:20625676

  18. Detection of Duchenne muscular dystrophy gene products in amniotic fluid and chorionic villus sampling cells.

    PubMed

    Prigojin, H; Brusel, M; Fuchs, O; Shomrat, R; Legum, C; Nudel, U; Yaffe, D

    1993-12-01

    We have examined the expression of several Duchenne muscular dystrophy (DMD) gene products in amniotic fluid (AF) and chorionic villus sampling (CVS) cells. Variable amounts of dystrophin could be detected in most CVS and AF samples by immunoprecipitation followed by Western blot analysis. PCR analysis demonstrated the presence of the muscle type dystrophin mRNA in all AF cell cultures. The brain type dystrophin mRNA was also detected in some of these cultures. These DMD gene transcripts are of fetal origin and are produced by most or all clonable AF cells. The results may facilitate the development of a method for prenatal diagnosis of DMD, based on the expression of the gene in AF and CVS cells. PMID:8253201

  19. Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid.

    PubMed

    Schläger, Christian; Körner, Henrike; Krueger, Martin; Vidoli, Stefano; Haberl, Michael; Mielke, Dorothee; Brylla, Elke; Issekutz, Thomas; Cabañas, Carlos; Nelson, Peter J; Ziemssen, Tjalf; Rohde, Veit; Bechmann, Ingo; Lodygin, Dmitri; Odoardi, Francesca; Flügel, Alexander

    2016-02-18

    In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen

  20. Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media

    PubMed Central

    Venna, Naresh Kumar; Murthy, Ch Lakshmi N.; Idris, Mohammed M.; Goel, Sandeep

    2015-01-01

    Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis) ocular fluid (BuOF) to replace FBS in cryomedia. Frozen–thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4), mouse embryonic stem (mES) cells, and primary cells, such as mouse embryonic fibroblast (MEF) cells, human peripheral blood mononuclear cells (hPBMCs), and mouse bone marrow cells (mBMCs), were cryopreserved in cryomedia containing 10% DMSO (D10) with 20% FBS (D10S20) or D10 with 20% BuOF (D10O20). For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen–thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA) proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types using

  1. Neural Differentiation of Human Umbilical Cord Mesenchymal Stem Cells by Cerebrospinal Fluid

    PubMed Central

    FARIVAR, Shirin; MOHAMADZADE, Zahra; SHIARI, Reza; FAHIMZAD, Alireza

    2015-01-01

    Objective Wharton’s jelly (WJ) is the gelatinous connective tissue from the umbilical cord. It is composed of mesenchymal stem cells, collagen fibers, and proteoglycans. The stem cells in WJ have properties that are interesting for research. For example, they are simple to harvest by noninvasive methods, provide large numbers of cells without risk to the donor, the stem cell population may be expanded in vitro, cryogenically stored, thawed, genetically manipulated, and differentiated in vitro. In our study, we investigated the effect of human cerebrospinal fluid (CSF) on neural differentiation of human WJ stem cells. Material & Methods The cells in passage 2 were induced into neural differentiation with different concentrations of human cerebrospinal fluid. Differentiation along with neural lineage was documented by expression of three neural markers: Nestin, Microtubule-Associated Protein 2 (MAP2), and Glial Fibrillary Astrocytic Protein (GFAP) for 21 days. The expression of the identified genes was confirmed by Reverse Transcriptase PCR (RT-PCR). Results Treatment with 100 and 200μg/ml CSF resulted in the expression of GFAP and glial cells marker on days 14 and 21. The expression of neural-specific genes following CSF treatment was dose-dependent and time-dependent. Treatment of the cells with a twofold concentration of CSF, led to the expression of MAP2 on day 14 of induction. No expression of GFAP was detected before day 14 or MAP2 before day 21, which shows the importance of the treatment period. In the present study, expression analysis for the known neural markers: Nestin, GFAP, and MAP2 using RT-PCR were performed. The data demonstrated that CSF could play a role as a strong inducer. Conclusion RT-PCR showed that cerebrospinal fluid promotes the expression of Nestin, MAP2, and GFAP mRNA in a dose-dependent manner, especially at a concentration of 200 μl/ml. In summary, CSF induces neurogenesis of WJ stem cells that encourages tissue engineering

  2. Effect of Embryonic Cerebrospinal Fluid on Proliferation and Differentiation of Neuroprogenitor Cells

    PubMed Central

    Yari, Siamak; Parivar, Kazem; Nabiuni, Mohammad; Keramatipour, Mohammad

    2013-01-01

    Objective: Embryonic cerebrospinal fluid (e-CSF) has an important role in development of embryonic and adult brain. Proteomic analysis suggests that this fluid has many morphogenes and cytokines that alter in time and space throughout embryonic life. The aim of this study was to evaluate the developmental effect of embryonic CSF on proliferation and differentiation of neuroprogenitor cells in different gestational age. Materials and Methods: In this In this experimental study, we examined the role of e- CSF on proliferation and differentiation of neuroprogenitor cells using neurosphere culture method. Neurospheres size analysis and MTT assay were used to assess cell proliferation after four days in vitro. Glial differentiation study was carried out by immunocytochemistry. Neurospheres size and percentage of glial fibrialy acidic protein (GFAP) positive cells were measured by image analyzer (image J). The data were analyzed by one-way ANOVA, followed by the Tukey’s post hoc test. Data were expressed as mean ± SEM, and differences were considered significant when p<0.05, 0.01 and 0.001. Results: Viability and proliferation of neuro progenitor cells in cultures conditioned with E16 CSF and E18 CSF were significantly increased compare to control group. A dramatic decrease in percentage of GFAP-positive cells was found following the application of CSF from E16 and E18 embryos, but not E20 CSF. Conclusion: Our data suggest that, e-CSF altered proliferation and differentiation of neuro progenitor cells in age dependent manner. E16 and E18 CSF enhanced proliferation and viability of neuro progenitor cells, and inhibited differentiation to glial fate in comparison with control group. PMID:23700558

  3. BOREAS AFM-06 Mean Temperature Profile Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from the National Oceanic and Atmospheric Adminsitration/Environment Technology Laboratory (NOAA/ETL) operated a 915-MHz wind/Radio Acoustic Sounding System (RASS) profiler system in the Southern Study Area (SSA) near the Old Jack Pine (OJP) tower from 21 May 1994 to 20 Sep 1994. The data set provides temperature profiles at 15 heights, containing the variables of virtual temperature, vertical velocity, the speed of sound, and w-bar. The data are stored in tabular ASCII files. The mean temperature profile data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  4. BOREAS AFM-6 Boundary Layer Height Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from National Oceanic and Atmospheric Adminsitration/Environment Technology Laboratory (NOAA/ETL) operated a 915-MHz wind/Radio Acoustic Sounding System (RASS) profiler system in the Southern Study Area (SSA) near the Old Jack Pine (OJP) site. This data set provides boundary layer height information over the site. The data were collected from 21 May 1994 to 20 Sep 1994 and are stored in tabular ASCII files. The boundary layer height data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  5. BOREAS AFM-06 Mean Wind Profile Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from the National Oceanic and Atmospheric Administration/Environment Technology Laboratory (NOAA/ETL) operated a 915-MHz wind/Radio Acoustic Sounding System (RASS) profiler system in the Southern Study Area (SSA) near the Old Jack Pine (OJP) tower from 21 May 1994 to 20 Sep 1994. The data set provides wind profiles at 38 heights, containing the variables of wind speed; wind direction; and the u-, v-, and w-components of the total wind. The data are stored in tabular ASCII files. The mean wind profile data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  6. [Differentiation of human amniotic fluid stem cells into cardiomyocytes through embryonic body formation].

    PubMed

    Wang, Han; Chen, Shuai; Cheng, Xiang; Dou, Zhongying; Wang, Huayan

    2008-09-01

    To isolate human amniotic fluid stem cells (hASCs) and induce hASCs into cardiomyocytes after forming the embryonic bodies. We cultivated hASCs isolated from the amniotic fluid continually for over 42 passages. The biological characteristics of hASCs were detected by immunocytochemistry, RT-PCR and flow cytometer, hASCs at 10-15th passage were suspension cultured to form embryonic bodies that were induced to cardiomyocytes. Fibroblastoid-type hASCs were obtained. Immunocytochemistry, RT-PCR and flow cytometry analysis demonstrated that hASCs were positive for some specific makers of the embryonic stem cell. hASCs could form embryonic bodies that were alkaline-phosphatase positive and expressed fgf5, zeta-globin and alpha-fetoprotein. The embryonic bodies could differentiate into cardiomyocytes showing alpha-actin positive and Tbx5, Nkx2.5, GATA4 and alpha-MHC positive. We conclued that hASCs obtained from human amniotic fluid could differentiate into cardiomyocytes through the formation of embryonic bodies. PMID:19160841

  7. Fuel cell assembly unit for promoting fluid service and electrical conductivity

    DOEpatents

    Jones, Daniel O.

    1999-01-01

    Fluid service and/or electrical conductivity for a fuel cell assembly is promoted. Open-faced flow channel(s) are formed in a flow field plate face, and extend in the flow field plate face between entry and exit fluid manifolds. A resilient gas diffusion layer is located between the flow field plate face and a membrane electrode assembly, fluidly serviced with the open-faced flow channel(s). The resilient gas diffusion layer is restrained against entering the open-faced flow channel(s) under a compressive force applied to the fuel cell assembly. In particular, a first side of a support member abuts the flow field plate face, and a second side of the support member abuts the resilient gas diffusion layer. The support member is formed with a plurality of openings extending between the first and second sides of the support member. In addition, a clamping pressure is maintained for an interface between the resilient gas diffusion layer and a portion of the membrane electrode assembly. Preferably, the support member is spikeless and/or substantially flat. Further, the support member is formed with an electrical path for conducting current between the resilient gas diffusion layer and position(s) on the flow field plate face.

  8. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

  9. Characteristics of silicone fluid as a pressure transmitting medium in diamond anvil cells

    NASA Astrophysics Data System (ADS)

    Shen, Yongrong; Kumar, Ravhi S.; Pravica, Michael; Nicol, Malcolm F.

    2004-11-01

    The properties of a silicone fluid with initial viscosity of 1 cst as a pressure transmitting medium for diamond anvil cells have been determined by ruby R1 line broadening and R1-R2 separation measurements to 64 GPa at ambient temperature. By these criteria, the silicone fluid is as good a pressure medium as a 4:1 methanol:ethanol mixture at low pressures to about 20 GPa, and is better than the mixture at higher pressures. Although argon media are better than the silicone at pressures to 30 GPa, this silicone behaves as well as argon at higher pressures. Furthermore, the silicone is easier to load than argon and is almost chemically inert.

  10. [Molecular Karyotyping of Cell-Free DNA from Blastocoele Fluid as a Basis for Noninvasive Preimplantation Genetic Screening of Aneuploidy].

    PubMed

    Skryabin, N A; Lebedev, I N; Artukhova, V G; Zhigalina, D I; Stepanov, I A; Krivoschekova, G V; Svetlakov, A V

    2015-11-01

    The discovery of DNA fragments in the blastocoele fluid is promising for the development of new noninvasive methods for the preimplantation genetic diagnosis of chromosomal diseases. However, to date there are no data confirming the concordance between the molecular karyotype of cell-free DNA from blastocoele fluid and the blastocyst cells per se. This paper reports on this concordance according to the results of molecular-cytogenetic analysis of the chromosomal set with the use of comparative genomic hybridization. PMID:26845860

  11. Amniotic fluid as a source of multipotent cells for clinical use.

    PubMed

    Young, Bruce K; Chan, Michael K; Liu, Li; Basch, Ross S

    2016-04-01

    Amniotic fluid cells (AFC) from 2nd trimester amniocentesis have been found to be a source of multipotent stem cells which might overcome the limitations of expansion, histocompatibility, tumorigenesis, and ethical issues associated with using human embryonic cells, umbilical cord, cord blood, bone marrow, and induced pluripotent cells. Previous work by our group and others demonstrated multipotency and the ability to grow well in culture. However, all these studies were done in media containing fetal calf serum. We sought to observe the properties of AFC grown in serum-free media as that would be required for clinical transplantation in humans. Fresh samples were obtained from three patients, and each sample divided into a culture whose cells were not exposed to fetal calf serum, and the other half into a standard culture medium containing fetal calf serum. Doubling time and stem cell marker expression by flow cytometry were assessed. Differentiation to neural, osteoid, and chondrogenic lineages was induced using appropriate media and confirmed by fluorescent microscopy, histology, and immunohistochemistry. There were no statistically significant differences between cells grown serum-free and in standard media in any of these parameters. The data supports the possibility of clinical use of AFC in stem cell transplantation. PMID:26115489

  12. Application of multiple levels of fluid shear stress to endothelial cells plated on polyacrylamide gels†

    PubMed Central

    Galie, P. A.; van Oosten, A.; Chen, C. S.

    2015-01-01

    Measurements of endothelial cell response to fluid shear stress have previously been performed on unphysiologically rigid substrates. We describe the design and implementation of a microfluidic device that applies discrete levels of shear stress to cells plated on hydrogel-based substrates of physiologicallyrelevant stiffness. The setup allows for measurements of cell morphology and inflammatory response to the combined stimuli, and identifies mechanisms by which vascular stiffening leads to pathological responses to blood flow. We found that the magnitude of shear stress required to affect endothelial cell morphology and inflammatory response depended on substrate stiffness. Endothelial cells on 100 Pa substrates demonstrate a greater increase in cell area and cortical stiffness and decrease in NF-κB nuclear translocation in response to TNF-α treatment compared to controls than cells plated on 10 kPa substrates. The response of endothelial cells on soft substrates to shear stress depends on the presence of hyaluronan (HA). These results emphasize the importance of substrate stiffness on endothelial function, and elucidate a means by which vascular stiffening in aging and disease can impact the endothelium. PMID:25573790

  13. Proton exchange membrane fuel cell degradation: A parametric analysis using Computational Fluid Dynamics

    NASA Astrophysics Data System (ADS)

    Ozden, Ender; Tari, Ilker

    2016-02-01

    A Polymer Electrolyte Membrane (PEM) fuel cell is numerically investigated both as fresh and as degraded with the help of observed degradation patterns reported in the literature. The fresh fuel cell model is validated and verified with the data from the literature. Modifying the model by varying the parameters affected by degradation, a degraded PEM fuel cell model is created. The degraded fuel cell is parametrically analyzed by using a commercial Computational Fluid Dynamics (CFD) software. The investigated parameters are the membrane equivalent weight, the Catalyst Layer (CL) porosity and viscous resistance, the Gas Diffusion Layer (GDL) porosity and viscous resistance, and the bipolar plate contact resistance. It is shown for the first time that PEM fuel cell overall degradation can be numerically estimated by combining experimental data from degraded individual components. By comparing the simulation results for the fresh and the degraded PEM fuel cells for two years of operation, it is concluded that the effects of overall degradation on cell potential is significant - estimated to be 17% around the operating point of the fuel cell at 0.95 V open circuit voltage and 70 °C operating temperature.

  14. In situ AFM crystal growth and dissolution study of calcite in the presence of aqueous fluoride

    NASA Astrophysics Data System (ADS)

    Vavouraki, A.; Putnis, C. V.; Putnis, A.; Koutsoukos, P. G.

    2009-04-01

    Fluoride is naturally abundant, encountered in rocks, soil and fresh and ocean water. Calcite crystals, during crystal growth may incorporate fluoride ions into their lattice (Okumura et al., 1983). In situ atomic force microscopy (AFM) has been used to study the growth and dissolution of calcite {104} surfaces in aqueous solutions in the presence of fluoride, using a fluid cell in which the supersaturated and the understaturated solutions respectively, flow over a freshly cleaved calcite crystal. For growth experiments, supersaturation index (S.I.) with respect to calcite was equal to 0.89 and the initial solution pH 10.2. The crystal growth rates were measured from the closure of the rhombohedral etch pits along the [010] direction induced by an initial dissolution step using pure water. The spreading rate of 2-dimensional nuclei was also measured along the same direction. In the presence of low fluoride concentrations (≤0.33 mM), the crystal growth rate of calcite was unaffected. At higher concentrations (up to 5 mM) growth rate decreased substantially to 50% of the rate in the absence of fluoride. Potential fluoride sorption over the calcite surface may ascribe the decrease of growth rates. Dissolution experiments were conducted at pH= 7.2 and dissolution rates of calcite were measured from the spreading of rhombohedral etch pits along both [010] and [42] directions. The presence of low concentrations of fluoride (≤1.1 mM) in the undersaturated solutions enhanced the dissolution rate along the [42] direction by 50% in comparison with pure water. The morphology of rhombohedral etch pits changed to hexagonal in the presence of fluoride in the undersaturated solutions. The AFM dissolution experiments suggested that the fluoride ions adsorbed onto the calcite surface. Further increase of fluoride concentrations (up to 1.6 mM) resulted in the decrease of the calcite dissolution rate by 60% in both [010] and [42] directions. Reference: Okumura, M, Kitano, Y

  15. A numerical investigation into the effects of fluid rheology and stroke kinematics on swimming alga cells in complex fluids

    NASA Astrophysics Data System (ADS)

    Li, Chuanbin; Guy, Robert; Thomases, Becca

    2015-11-01

    It is observed in experiments that when the fluid viscosity or elasticity is changed, Chlamydomonas reinhardtii exhibits changes in both flagellar kinematics and the swimming speed. In order to understand the effects of rheology on both gait and swimming performance, we develop a computational model of the swimmer. We use flagellar strokes fit from experimental data to set up a constrained system, determining the forces on the swimmer and its swimming velocity. Our approach to simulating the swimming behavior demonstrates low computational costs even in three dimensions. In our simulations, stroke patterns and fluid rheologies are changed separately, so that we can dissect the contributions of stroke kinematics of the alga and the fluid environment, which can not be achieved with experiments.

  16. Human amniotic fluid stem cell injection therapy for urethral sphincter regeneration in an animal model

    PubMed Central

    2012-01-01

    Background Stem cell injection therapies have been proposed to overcome the limited efficacy and adverse reactions of bulking agents. However, most have significant limitations, including painful procurement, requirement for anesthesia, donor site infection and a frequently low cell yield. Recently, human amniotic fluid stem cells (hAFSCs) have been proposed as an ideal cell therapy source. In this study, we investigated whether periurethral injection of hAFSCs can restore urethral sphincter competency in a mouse model. Methods Amniotic fluids were collected and harvested cells were analyzed for stem cell characteristics and in vitro myogenic differentiation potency. Mice underwent bilateral pudendal nerve transection to generate a stress urinary incontinence (SUI) model and received either periurethral injection of hAFSCs, periurethral injection of Plasma-Lyte (control group), or underwent a sham (normal control group). For in vivo cell tracking, cells were labeled with silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate (MNPs@SiO2 (RITC)) and were injected into the urethral sphincter region (n = 9). Signals were detected by optical imaging. Leak point pressure and closing pressure were recorded serially after injection. Tumorigenicity of hAFSCs was evaluated by implanting hAFSCs into the subcapsular space of the kidney, followed two weeks later by retrieval and histologic analysis. Results Flow activated cell sorting showed that hAFSCs expressed mesenchymal stem cell (MSC) markers, but no hematopoietic stem cell markers. Induction of myogenic differentiation in the hAFSCs resulted in expression of PAX7 and MYOD at Day 3, and DYSTROPHIN at Day 7. The nanoparticle-labeled hAFSCs could be tracked in vivo with optical imaging for up to 10 days after injection. Four weeks after injection, the mean LPP and CP were significantly increased in the hAFSC-injected group compared with the control group. Nerve regeneration and neuromuscular junction

  17. Effect of Tamarindus indica L. leaves' fluid extract on human blood cells.

    PubMed

    Escalona-Arranz, J C; Garcia-Diaz, J; Perez-Rosés, R; De la Vega, J; Rodríguez-Amado, J; Morris-Quevedo, H J

    2014-01-01

    Tamarind leaves are edible; however, their saponin content could be toxic to human blood cells. In this article, the effect of tamarind leaf fluid extract (TFE) on human blood cells was evaluated by using several tests. Results revealed that TFE did not cause significant haemolysis on human red blood cells even at the lowest evaluated concentration (20 mg/mL). Blood protein denaturalisation ratio was consistently lower than in control at TFE concentrations greater than 40 mg/mL. Erythrocyte membrane damage caused by the action of oxidative H2O2 displayed a steady reduction with increasing TFE concentrations. In the reactive oxygen species (ROS) measurement by using flow cytometry assay, leucocyte viability was over 95% at tested concentrations, and a high ROS inhibition was also recorded. Protective behaviour found in TFE should be attributed to its polyphenol content. Thus, tamarind leaves can be regarded as a potential source of interesting phytochemicals. PMID:24773365

  18. Cell Attachment Behavior on Solid and Fluid Substrates Exhibiting Spatial Patterns of Physical Properties

    PubMed Central

    Oliver, Ann E.; Ngassam, Viviane; Dang, Phuong; Sanii, Babak; Wu, Huawen; Yee, Chanel K.; Yeh, Yin; Parikh, Atul N.

    2009-01-01

    The ability to direct proliferation and growth of living cells using chemically and topologically textured surfaces is finding many niche applications, both in fundamental biophysical investigations of cell-surface attachment as well as in developing design principles for many tissue engineering applications. Here we address cellular adhesion behavior on solid patterns of differing wettability (a static substrate) and fluid patterns of membrane topology (a dynamic substrate). We find striking differences in the cellular adhesion characteristics of lipid mono- and bilayers, despite their essentially identical surface chemical and structural character. These differences point to the importance of subtle variations in the physical properties of the lipid mono- and bilayers (e.g., membrane tension and out-of-plane undulations). Furthermore, we find that introducing phosphatidylserine into the patterned lipidic substrates causes a loss of cell-patterning capability. Implications of this finding for the mechanism by which phosphatidylserine promotes cellular adhesion are discussed. PMID:19453187

  19. Enabling the measurement of in-situ, atomic scale mineral transformation rates in supercritical CO2 through development of a high pressure AFM

    NASA Astrophysics Data System (ADS)

    Lea, S.; Higgins, S. R.; Knauss, K. G.; Rosso, K. M.

    2010-12-01

    Capture and storage of carbon dioxide in deep geologic formations represents one promising scenario for minimizing the impacts of greenhouse gases on global warming. The ability to demonstrate that CO2 will remain stored in the geological formation over the long-term is needed in support of widespread implementation decisions, and knowledge of mineral-fluid chemical transformation rates is an essential aspect. The majority of previous research on mineral-fluid interactions has focused primarily on the reactivity of minerals in aqueous solutions containing various amounts of dissolved CO2. Long-term caprock integrity, however, could also be dictated by mineral transformations occurring in low-water environments dominated by the supercritical CO2 (scCO2) fluid phase, which is expected to slowly displace or dessicate residual aqueous solution at the caprock-fluid interface. Many of the mechanisms of mineral interfacial reactions with hydrated or water-saturated scCO2 are unknown and there are unique challenges to obtain kinetic and thermodynamic data for mineral transformation reactions in these fluids. We are developing a high-pressure atomic force microscope (AFM) that will enable in-situ, atomic scale measurements of metal carbonate nucleation and growth rates on mineral surfaces in contact with hydrated scCO2 fluids. This apparatus is based on the hydrothermal AFM that was developed by Higgins et al.1, but includes some enhancements and is designed to handle pressures up to 100 bar. The noise in our optically-based cantilever deflection detection scheme is subject to perturbations in the density (due to index of refraction dependence) of the compressible supercritical fluid. Consequently, variations in temperature and pressure within the fluid cell are a primary technical challenge with possible significant impact in imaging resolution. We demonstrate with our test fluid cell that the equivalent rms noise in the deflection signal is similar to (and in some cases

  20. Defect in recruiting effector memory CD8+ T-cells in malignant pleural effusions compared to normal pleural fluid

    PubMed Central

    2013-01-01

    Background Malignant pleural effusions (MPE) are a common and fatal complication in cancers including lung or breast cancers, or malignant pleural mesothelioma (MPM). MPE animal models and immunotherapy trials in MPM patients previously suggested defects of the cellular immunity in MPE. However only few observational studies of the immune response were done in MPM patients, using questionable control groups (transudate…). Methods We compared T cell populations evaluated by flow cytometry from blood and pleural effusion of untreated patients with MPM (n = 58), pleural metastasis of adenocarcinoma (n = 30) or with benign pleural lesions associated with asbestos exposure (n = 23). Blood and pleural fluid were also obtained from healthy subjects, providing normal values for T cell populations. Results Blood CD4+ or CD8+ T cells percentages were similar in all groups of patients or healthy subjects. Whereas pleural fluid from healthy controls contained mainly CD8+ T cells, benign or malignant pleural effusions included mainly CD4+ T cells. Effector memory T cells were the main T cell subpopulation in pleural fluid from healthy subjects. In contrast, there was a striking and selective recruitment of central memory CD4+ T cells in MPE, but not of effector cells CD8+ T cells or NK cells in the pleural fluid as one would expect in order to obtain an efficient immune response. Conclusions Comparing for the first time MPE to pleural fluid from healthy subjects, we found a local defect in recruiting effector CD8+ T cells, which may be involved in the escape of tumor cells from immune response. Further studies are needed to characterize which subtypes of effector CD8+ T cells are involved, opening prospects for cell therapy in MPE and MPM. PMID:23816056

  1. Fluid Shear Stress Regulates the Invasive Potential of Glioma Cells via Modulation of Migratory Activity and Matrix Metalloproteinase Expression

    PubMed Central

    Qazi, Henry; Shi, Zhong-Dong; Tarbell, John M.

    2011-01-01

    Background Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate. Methodology/Principal Findings A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs. Conclusions/Significance Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression. PMID:21637818

  2. Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway.

    PubMed

    Chang, Jessica T; Lehtinen, Maria K; Sive, Hazel

    2016-01-01

    Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss-of-function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival. PMID:25980532

  3. Fluctuations of coupled fluid and solid membranes with application to red blood cells.

    PubMed

    Auth, Thorsten; Safran, S A; Gov, Nir S

    2007-11-01

    The fluctuation spectra and the intermembrane interaction of two membranes at a fixed average distance are investigated. Each membrane can either be a fluid or a solid membrane, and in isolation, its fluctuations are described by a bare or a wave-vector-dependent bending modulus, respectively. The membranes interact via their excluded-volume interaction; the average distance is maintained by an external, homogeneous pressure. For strong coupling, the fluctuations can be described by a single, effective membrane that combines the elastic properties. For weak coupling, the fluctuations of the individual, noninteracting membranes are recovered. The case of a composite membrane consisting of one fluid and one solid membrane can serve as a microscopic model for the plasma membrane and cytoskeleton of the red blood cell. We find that, despite the complex microstructure of bilayers and cytoskeletons in a real cell, the fluctuations with wavelengths lambda greater, similar 400 nm are well described by the fluctuations of a single, polymerized membrane (provided that there are no inhomogeneities of the microstructure). The model is applied to the fluctuation data of discocytes ("normal" red blood cells), a stomatocyte, and an echinocyte. The elastic parameters of the membrane and an effective temperature that quantifies active, metabolically driven fluctuations are extracted from the experiments. PMID:18233690

  4. Fluctuations of coupled fluid and solid membranes with application to red blood cells

    NASA Astrophysics Data System (ADS)

    Auth, Thorsten; Safran, S. A.; Gov, Nir S.

    2007-11-01

    The fluctuation spectra and the intermembrane interaction of two membranes at a fixed average distance are investigated. Each membrane can either be a fluid or a solid membrane, and in isolation, its fluctuations are described by a bare or a wave-vector-dependent bending modulus, respectively. The membranes interact via their excluded-volume interaction; the average distance is maintained by an external, homogeneous pressure. For strong coupling, the fluctuations can be described by a single, effective membrane that combines the elastic properties. For weak coupling, the fluctuations of the individual, noninteracting membranes are recovered. The case of a composite membrane consisting of one fluid and one solid membrane can serve as a microscopic model for the plasma membrane and cytoskeleton of the red blood cell. We find that, despite the complex microstructure of bilayers and cytoskeletons in a real cell, the fluctuations with wavelengths λ≳400nm are well described by the fluctuations of a single, polymerized membrane (provided that there are no inhomogeneities of the microstructure). The model is applied to the fluctuation data of discocytes (“normal” red blood cells), a stomatocyte, and an echinocyte. The elastic parameters of the membrane and an effective temperature that quantifies active, metabolically driven fluctuations are extracted from the experiments.

  5. Acoustic micro-vortexing of fluids, particles and cells in disposable microfluidic chips.

    PubMed

    Iranmanesh, Ida; Ohlin, Mathias; Ramachandraiah, Harisha; Ye, Simon; Russom, Aman; Wiklund, Martin

    2016-08-01

    We demonstrate an acoustic platform for micro-vortexing in disposable polymer microfluidic chips with small-volume (20 μl) reaction chambers. The described method is demonstrated for a variety of standard vortexing functions, including mixing of fluids, re-suspension of a pellet of magnetic beads collected by a magnet placed on the chip, and lysis of cells for DNA extraction. The device is based on a modified Langevin-type ultrasonic transducer with an exponential horn for efficient coupling into the microfluidic chip, which is actuated by a low-cost fixed-frequency electronic driver board. The transducer is optimized by numerical modelling, and different demonstrated vortexing functions are realized by actuating the transducer for varying times; from fractions of a second for fluid mixing, to half a minute for cell lysis and DNA extraction. The platform can be operated during 1 min below physiological temperatures with the help of a PC fan, a Peltier element and an aluminum heat sink acting as the chip holder. As a proof of principle for sample preparation applications, we demonstrate on-chip cell lysis and DNA extraction within 25 s. The method is of interest for automating and chip-integrating sample preparation procedures in various biological assays. PMID:27444649

  6. Membrane with internal passages to permit fluid flow and an electrochemical cell containing the same

    NASA Technical Reports Server (NTRS)

    Cisar, Alan J. (Inventor); Gonzalez-Martin, Anuncia (Inventor); Hitchens, G. Duncan (Inventor); Murphy, Oliver J. (Inventor)

    1997-01-01

    The invention provides an improved proton exchange membrane for use in electrochemical cells having internal passages parallel to the membrane surface, an apparatus and process for making the membrane, membrane and electrode assemblies fabricated using the membrane, and the application of the membrane and electrode assemblies to a variety of devices, both electrochemical and otherwise. The passages in the membrane extend from one edge of the membrane to another and allow fluid flow through the membrane and give access directly to the membrane for purposes of hydration.

  7. Case Report: Detection and quantification of tumor cells in peripheral blood and ascitic fluid from a metastatic esophageal cancer patient using the CellSearch (®) technology.

    PubMed

    Tu, Qian; Bittencourt, Marcelo De Carvalho; Cai, Huili; Bastien, Claire; Lemarie-Delaunay, Camille; Bene, Marie C; Faure, Gilbert C

    2014-01-01

    Analysis of ascitic fluid should help to identify and characterize malignant cells in gastrointestinal cancer. However, despite a high specificity, the sensitivity of traditional ascitic fluid cytology remains insufficient, at around 60%. Since 2004 the CellSearch (®) technology has shown its advantages in the detection of circulating tumor cells (CTCs) in peripheral blood, which can perform an accurate diagnosis and molecular analysis at the same time. To our knowledge, no previous study has explored the potential utility of this technology for the detection and quantification of tumor cells in ascitic fluid samples. Herein we report a case of metastatic esophageal adenocarcinoma in a 70-year-old man presenting with dysphagia and a large amount of fluid in the peritoneal cavity. Analysis of a peripheral blood sample and ascites sample with the CellSearch (®) technology both revealed the presence of putative tumor cells that were positive for epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) expression. This study confirmed the hematogenous dissemination of esophageal cancer by the detection of circulating tumor cells in the peripheral blood, and is the first to demonstrate that tumor cells can be identified in ascitic fluid by using CellSearch (®) technology. PMID:25075284

  8. Can Outer Hair Cells Actively Pump Fluid into the Tunnel of Corti?

    NASA Astrophysics Data System (ADS)

    Zagadou, Brissi Franck; Mountain, David C.

    2011-11-01

    Non-classical models of the cochlear traveling wave have been introduced in attempt to capture the unique features of the cochlear amplifier (CA). These models include multiple modes of longitudinal coupling. In one approach, it is hypothesized that two wave modes can add their energies to create amplification such as that desired in the CA. The tunnel of Corti (ToC) was later used to represent the second wave mode for the proposed traveling wave amplifier model, and was incorporated in a multi-compartment cochlea model. The results led to the hypothesis that the CA functions as a fluid pump. However, this hypothesis must be consistent with the anatomical structure of the organ of Corti (OC). The fluid must pass between the outer pillar cells before reaching the ToC, and the ToC fluid and the underlying basilar membrane must constitute an appropriate waveguide. We have analyzed an anatomically based 3D finite element model of the ToC of the gerbil. Our results demonstrate that the OC structure is consistent with the hypothesis.

  9. Delayed antibody synthesis in mice after transfer of immune peritoneal fluid cells

    PubMed Central

    Weiler, E.

    1964-01-01

    Ascites fluid, rich in bacteriophage-neutralizing antibody, was produced when mice were treated first, with lethal or near-lethal whole body X-radiation; secondly, intravenous injection of spleen cells from donor mice immunized against bacteriophage; and thirdly, with an intraperitoneal injection of bacteriophage in Freund's adjuvant. The `immune ascites cells' were washed and transferred to other mice without further addition of antigen. The production of phage-neutralizing antibody in recipient mice showed the following properties. (1) The highest rate of antibody synthesis occurred between the 5th and the 11th day after cell transfer. In contrast, spleen cells similarly transferred gave rise to antibody formation with the maximum rate of synthesis immediately after transfer. (2) The antibody formation occurred essentially only in isologous recipients, not in homologous ones, whether the latter were pre-immunized against cells of the donor strain or not. With spleen cells, antibody synthesis was not impaired in homologous hosts for about 4 days after transfer, if the hosts were not pre-immunized against the donor strain. (3) Freezing and thawing of the donor cells prior to injection into the hosts abolished subsequent antibody synthesis. (4) Irradiation of the cells with 650 R. abolished antibody formation after transfer. (5) Whole-body irradiation of the recipient mice resulted in increased antibody formation. (6) When immune ascites cells were injected into newborn mice, high levels of antibody were found 13 days afterwards. It is concluded (a) that the population of immune ascites cells carries both the specific information and the stimulus for antibody synthesis, and (b) that the antibody-forming apparatus is not yet present in a functional state at the time of transfer, but develops several days afterwards in the host mice. PMID:14169104

  10. Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction

    NASA Astrophysics Data System (ADS)

    Bethel, Kelly; Luttgen, Madelyn S.; Damani, Samir; Kolatkar, Anand; Lamy, Rachelle; Sabouri-Ghomi, Mohsen; Topol, Sarah; Topol, Eric J.; Kuhn, Peter

    2014-02-01

    Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay), to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs. Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined by positive staining for DAPI, CD146 and von Willebrand Factor and negative staining for CD45. In addition, CECs exhibited distinct morphological features that enable differentiation from surrounding white blood cells. CECs were found both as individual cells and as aggregates. CEC numbers were higher in MI patients compared with healthy controls. VS patients had lower CEC counts when compared with MI patients but were not different from healthy controls. Both HD-CEC and CellSearch® assays could discriminate MI patients from healthy controls with comparable accuracy but the HD-CEC assay exhibited higher specificity while maintaining high sensitivity. Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

  11. Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow.

    PubMed

    Orhon, Idil; Dupont, Nicolas; Zaidan, Mohamad; Boitez, Valérie; Burtin, Martine; Schmitt, Alain; Capiod, Thierry; Viau, Amandine; Beau, Isabelle; Wolfgang Kuehn, E; Friedlander, Gérard; Terzi, Fabiola; Codogno, Patrice

    2016-06-01

    Autophagy is an adaptation mechanism that is vital for cellular homeostasis in response to various stress conditions. Previous reports indicate that there is a functional interaction between the primary cilium (PC) and autophagy. The PC, a microtubule-based structure present at the surface of numerous cell types, is a mechanical sensor. Here we show that autophagy induced by fluid flow regulates kidney epithelial cell volume in vitro and in vivo. PC ablation blocked autophagy induction and cell-volume regulation. In addition, inhibition of autophagy in ciliated cells impaired the flow-dependent regulation of cell volume. PC-dependent autophagy can be triggered either by mTOR inhibition or a mechanism dependent on the polycystin 2 channel. Only the LKB1-AMPK-mTOR signalling pathway was required for the flow-dependent regulation of cell volume by autophagy. These findings suggest that therapies regulating autophagy should be considered in developing treatments for PC-related diseases. PMID:27214279

  12. Particle deformation induced by AFM tapping under different setpoint voltages

    NASA Astrophysics Data System (ADS)

    Wu, Chung-Lin; Farkas, Natalia; Dagata, John A.; He, Bo-Ching; Fu, Wei-En

    2014-09-01

    The measured height of polystyrene nanoparticles varies with setpoint voltage during atomic force microscopy (AFM) tapping-mode imaging. Nanoparticle height was strongly influenced by the magnitude of the deformation caused by the AFM tapping forces, which was determined by the setpoint voltage. This influence quantity was studied by controlling the operational AFM setpoint voltage. A test sample consisting of well-dispersed 60-nm polystyrene and gold nanoparticles co-adsorbed on poly-l-lysine-coated mica was studied in this research. Gold nanoparticles have not only better mechanical property than polystyrene nanoparticles, but also obvious facets in AFM phase image. By using this sample of mixed nanoparticles, it allows us to confirm that the deformation resulted from the effect of setpoint voltage, not noise. In tapping mode, the deformation of polystyrene nanoparticles increased with decreasing setpoint voltage. Similar behavior was observed with both open loop and closed loop AFM instruments.

  13. Combination of fluid and solid mechanical stresses contribute to cell death and detachment in a microfluidic alveolar model.

    PubMed

    Douville, Nicholas J; Zamankhan, Parsa; Tung, Yi-Chung; Li, Ran; Vaughan, Benjamin L; Tai, Cheng-Feng; White, Joshua; Christensen, Paul J; Grotberg, James B; Takayama, Shuichi

    2011-02-21

    Studies using this micro-system demonstrated significant morphological differences between alveolar epithelial cells (transformed human alveolar epithelial cell line, A549 and primary murine alveolar epithelial cells, AECs) exposed to combination of solid mechanical and surface-tension stresses (cyclic propagation of air-liquid interface and wall stretch) compared to cell populations exposed solely to cyclic stretch. We have also measured significant differences in both cell death and cell detachment rates in cell monolayers experiencing combination of stresses. This research describes new tools for studying the combined effects of fluid mechanical and solid mechanical stress on alveolar cells. It also highlights the role that surface tension forces may play in the development of clinical pathology, especially under conditions of surfactant dysfunction. The results support the need for further research and improved understanding on techniques to reduce and eliminate fluid stresses in clinical settings. PMID:21152526

  14. Bioimpedance spectroscopy for clinical assessment of fluid distribution and body cell mass.

    PubMed

    Earthman, Carrie; Traughber, Diana; Dobratz, Jennifer; Howell, Wanda

    2007-08-01

    Body composition assessment has been used to evaluate clinical interventions in research trials, and has the potential to improve patient care in the clinical setting. Body cell mass (BCM) is an important indicator of nutrition status; however, its measurement in the clinic has been limited. BCM can be estimated by the measurement of intracellular water (ICW). The assessment of extracellular water (ECW) is also important because many clinical populations undergo alterations in fluid distribution, particularly individuals with wasting, those receiving dialysis, and obese individuals. Bioimpedance spectroscopy (BIS) is a unique bioimpedance approach that differs in underlying basis from the more readily recognized single-frequency bioelectrical impedance analysis (SF-BIA) in that it does not require the use of statistically derived, population-specific prediction equations. It has the potential advantage of not only measuring total body water (TBW), as does SF-BIA, but also offering the unique capacity to differentiate between ECW and ICW and, thus, to provide an estimate of BCM. This literature review was conducted to compare available BIS devices to multiple dilution for measuring fluid compartments or BCM in a number of populations. Variable results regarding the ability of BIS to measure absolute volumes, as well as the observation of wide limits of variation, make BIS problematic for individual assessment in the clinic, particularly in populations with abnormal fluid distribution or body geometry. BIS has been found to be more accurate for measuring changes in fluid volumes or BCM, particularly in post-surgical and human immunodeficiency virus (HIV)-infected individuals. It is certainly possible that population-specific adjustments may improve the accuracy of BIS for assessing individuals in the clinical setting; however, additional research and development is needed before the method can be accepted for routine clinical use. PMID:17644693

  15. Fluid balance of pediatric hematopoietic stem cell transplant recipients and intensive care unit admission.

    PubMed

    Benoit, Geneviève; Phan, Véronique; Duval, Michel; Champagne, Martin; Litalien, Catherine; Merouani, Aicha

    2007-03-01

    Fluid administration is essential in patients undergoing hematopoietic stem cell transplant (HSCT). Admission to pediatric intensive care unit (PICU) is required for 11-29% of pediatric HSCT recipients and is associated with high mortality. The objective of this study was to determine if a positive fluid balance acquired during the HSCT procedure is a risk factor for PICU admission. The medical records of 87 consecutive children who underwent a first HSCT were reviewed retrospectively for the following periods: from admission for HSCT to PICU admission for the first group (PICU group), and from admission for HSCT to hospital discharge for the second group (non-PICU group). Fluid balance was determined on the basis of weight gain (WG) and fluid overload (FO). PICU group consisted of 19 patients (21.8%). Among these, 13 (68.4%) developed>or=10% WG prior to PICU admission compared with 15 (22.1%) in the non-PICU group (p<0.001). Thirteen patients (68.4%) developed>or=10% FO prior to PICU admission compared with 31 (45.6%) in the non-PICU group (p=0.075). Following multivariate analysis, >or=10% WG (p=0.018) and cardiac dysfunction on admission for HSCT (p=0.036) remained independent risk factors for PICU admission. Smaller children (p=0.033) and patients with a twofold increase in serum creatinine (p=0.026) were at risk of developing>or=10% WG. This study shows that WG is a risk factor for PICU admission in pediatric HSCT recipients. Further research is needed to better understand the pathophysiology of WG in these patients and to determine the impact of WG prevention on PICU admission. PMID:17123119

  16. BOREAS AFM-07 SRC Surface Meteorological Data

    NASA Technical Reports Server (NTRS)

    Osborne, Heather; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Young, Kim; Wittrock, Virginia; Shewchuck, Stan; Smith, David E. (Technical Monitor)

    2000-01-01

    The Saskatchewan Research Council (SRC) collected surface meteorological and radiation data from December 1993 until December 1996. The data set comprises Suite A (meteorological and energy balance measurements) and Suite B (diffuse solar and longwave measurements) components. Suite A measurements were taken at each of ten sites, and Suite B measurements were made at five of the Suite A sites. The data cover an approximate area of 500 km (North-South) by 1000 km (East-West) (a large portion of northern Manitoba and northern Saskatchewan). The measurement network was designed to provide researchers with a sufficient record of near-surface meteorological and radiation measurements. The data are provided in tabular ASCII files, and were collected by Aircraft Flux and Meteorology (AFM)-7. The surface meteorological and radiation data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  17. L-selectin-mediated lymphocyte-cancer cell interactions under low fluid shear conditions.

    PubMed

    Resto, Vicente A; Burdick, Monica M; Dagia, Nilesh M; McCammon, Susan D; Fennewald, Susan M; Sackstein, Robert

    2008-06-01

    Cell migration in blood flow is mediated by engagement of specialized adhesion molecules that function under hemodynamic shear conditions, and many of the effectors of these adhesive interactions, such as the selectins and their ligands, are well defined. However, in contrast, our knowledge of the adhesion molecules operant under lymphatic flow conditions is incomplete. Among human malignancies, head and neck squamous cell cancer displays a marked predilection for locoregional lymph node metastasis. Based on this distinct tropism, we hypothesized that these cells express adhesion molecules that promote their binding to lymphoid tissue under lymphatic fluid shear stress. Accordingly, we investigated adhesive interactions between these and other cancer cells and the principal resident cells of lymphoid organs, lymphocytes. Parallel plate flow chamber studies under defined shear conditions, together with biochemical analyses, showed that human head and neck squamous cell cancer cells express heretofore unrecognized L-selectin ligand(s) that mediate binding to lymphocyte L-selectin at conspicuously low shear stress levels of 0.07-0.08 dynes/cm(2), consistent with lymphatic flow. The binding of head and neck squamous cancer cells to L-selectin displays canonical biochemical features, such as requirements for sialylation, sulfation, and N-glycosylation, but displays a novel operational shear threshold differing from all other L-selectin ligands, including those expressed on colon cancer and leukemic cells (e.g. HCELL). These data define a novel class of L-selectin ligands and expand the scope of function for L-selectin within circulatory systems to now include a novel activity within shear stresses characteristic of lymphatic flow. PMID:18385135

  18. Immunophenotypic analysis of cerebrospinal fluid cell populations with the Cell-Dyn Sapphire haematology analyser: method feasibility and preliminary observations.

    PubMed

    Adam, P; Sobek, O; Scott, C S; Dolezil, D; Kasik, J; Hajdukova, L; Adam, D

    2010-02-01

    Cerebrospinal fluid (CSF) samples (n=50) from patients with neurological disease (bacterial infection, viral infection, neuroborreliosis and multiple sclerosis) were analysed to characterize cell populations by fluorescent immunocytometry with the CD-Sapphire haematology analyser. Reagent combinations applied to all CSF samples comprised CD3/CD19/HLA-DR and CD4/CD8, with some being further analysed using CD3/CD4, CD3/CD16 and CD3/CD25 protocols. Of the 50 samples, 11 were excluded because of high proportions of nonviable cells (n=2) or insufficient cell numbers (n=9). Apart from bacterial infection with granulocytosis, all diagnostic groups showed high proportions (51.4-77.0%) of CD3+ T cells. There was a modest association between T-cell and B-cell counts, but absolute B-cell numbers exceeded 5 cells/microl in only 7/39 cases (neuroborreliosis, n=6; bacterial meningitis, n=1). CD3/Ia antigen (activation) co-expression was low and only exceeded 5% in 7/39 samples with no diagnostic correlation. Primary CD4+ and CD8+ T-cell subsets showed similar quantitative trends and CD4/CD8 co-analysis revealed the presence in all diagnostic groups (neuroborreliosis and multiple sclerosis in particular) of a CD4+CD8int fraction that was predominantly CD3+ and CD16- and had a morphological profile consistent with small lymphoid cells. Supplementary CD-Sapphire cellular immunological analysis of most CSF samples is feasible using the procedure detailed in this communication. PMID:19500178

  19. The effect of wound fluid on adipose-derived stem cells in vitro: a study in human cell materials.

    PubMed

    Scherzed, Agmal; Hackenberg, Stephan; Froelich, Katrin; Radeloff, Andreas; Technau, Antje; Kessler, Michael; Hagen, Rudolf; Rak, Kristen; Koehler, Christian; Kleinsasser, Norbert

    2011-08-01

    After surgery, wound healing begins with a well-orchestrated integration of several cytokines, cells, and extracellular matrix. Some studies show an involvement of stem cells in wound healing. However, little is known about the mechanism that leads to the migration of stem cells. Wound fluid (WF) with its cytokines may play an important role. We investigated in the present study the in vitro effects of WF on adipose-derived stem cells (ADSCs). Survival, proliferation, structural integrity, changes in the multidifferentiation potential, and surface markers (cluster of differentiation [CD] 105, CD73, CD90) of ADSCs after cultivation with WF was analyzed. Further, the migration effect of WF on ADSCs was evaluated. The proliferation rate and the migration potential of ADSCs were enhanced significantly by cultivation with WF. There was also a change in the quantity of surface markers after cultivation with WF. In conclusion, in vitro expansion of stem cells with WF proved possible. WF and its cytokines could represent one primary reason for the migration of stem cells toward the wound. Future investigation is warranted to clarify the significance of the shift in surface markers. PMID:21457100

  20. Indications for near-surface fluid circulation cells at bacterial mats

    NASA Astrophysics Data System (ADS)

    Gubsch, S.; Haeckel, M.; Wallmann, K.

    2009-04-01

    At submarine cold vents off Costa Rica detailed sediment sampling along transects across bacterial mats was conducted during expedition M66/2 with RV METEOR deploying a remotely operated vehicle (ROV). Bacterial mats occurred in patches of several m2 in size covering the sediment surface. Porewater analyses of the pushcore sediments revealed rapid sulfate consumption due to anaerobic methane oxidation (AMO) below the bacterial mats. SO4 was depleted at ~5 cm sediment depth in the center of the mat and penetrating deeper into the sediment towards the rim of the mat. Pushcores taken in the center of these mats, however, showed a subsequent increase of sulfate concentrations below a sediment depth of ~10 cm. Other dissolved compounds, such as Cl, Br, H2S, TA, NH4, PO4, and SiO4, showed a similar behaviour with concentrations returning towards bottomwater values. Since this trend is common to all of the solutes, it is most likely explained by a physical process. We assume that focussed fluid outflow near the center of the bacterial mat creates a convective flow cell with bottom waters penetrating into the adjacent sediment area and directed towards the flow channel. A set of different 2-D and 3-D transport-reaction models were developed to test this hypothesis. Fluid flow in the central channel turned out to be homogeneous and thus, could be resembled as boundary condition of the surrounding sediment domain. The model also includes AMO as the most important reaction of a cold vent system. Model results indicate that the observed porewater sulfate and chloride profiles can be reproduced fairly well, for example, when applying an advection velocity of 100 cm/a in the central fluid channel and a mean background advection of 3 cm/a in the sediment domain. A detailed sensitivity study has been performed determining the parameters dominating the establishment of the near-surface flow cell.

  1. Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids

    PubMed Central

    Song, Yeonhwa; Yun, Sujin; Yang, Hye Jin; Yoon, A Young; Kim, Haekwon

    2012-01-01

    Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×104 as the least for the low density group, and 29.3±2.8×104 as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids. PMID:25949109

  2. Isolation of Human Neural Stem Cells from the Amniotic Fluid with Diagnosed Neural Tube Defects.

    PubMed

    Chang, Yu-Jen; Su, Hong-Lin; Hsu, Lee-Feng; Huang, Po-Jui; Wang, Tzu-Hao; Cheng, Fu-Chou; Hsu, Li-Wen; Tsai, Ming-Song; Chen, Chih-Ping; Chang, Yao-Lung; Chao, An-Shine; Hwang, Shiaw-Min

    2015-08-01

    Human neural stem cells (NSCs) are particularly valuable for the study of neurogenesis process and have a therapeutic potential in treating neurodegenerative disorders. However, current progress in the use of human NSCs is limited due to the available NSC sources and the complicated isolation and culture techniques. In this study, we describe an efficient method to isolate and propagate human NSCs from the amniotic fluid with diagnosed neural tube defects (NTDs), specifically, anencephaly. These amniotic fluid-derived NSCs (AF-NSCs) formed neurospheres and underwent long-term expansion in vitro. In addition, these cells showed normal karyotypes and telomerase activity and expressed NSC-specific markers, including Nestin, Sox2, Musashi-1, and the ATP-binding cassette G2 (ABCG2). AF-NSCs displayed typical morphological patterns and expressed specific markers that were consistent with neurons, astrocytes, oligodendrocytes, and dopaminergic neurons after proper induction conditions. Furthermore, grafted AF-NSCs improved the physiological functions in a rat stroke model. The ability to isolate and bank human NSCs from this novel source provides a unique opportunity for translational studies of neurological disorders. PMID:25923707

  3. Biotechnological promises of Fe-filled CNTs for cell shepherding and magnetic fluid hyperthermia applications

    NASA Astrophysics Data System (ADS)

    Pineux, Florent; Marega, Riccardo; Stopin, Antoine; La Torre, Alessandro; Garcia, Yann; Devlin, Eamonn; Michiels, Carine; N. Khlobystov, Andrei; Bonifazi, Davide

    2015-12-01

    Fe-filled carbon nanotubes (Fe@CNTs) recently emerged as an effective class of hybrid nanoparticles for biotechnological applications, such as magnetic cell sorting and magnetic fluid hyperthermia. Aiming at studying the effects of both the Fe loading and the magnetocrystalline characteristics in these applications, we describe herein the preparation of Fe@CNTs containing different Fe phases that, upon functionalization with the antibody Cetuximab (Ctxb), allow the targeting of cancer cells. Our experimental findings reveal that an optimal Ctxb/Fe weight ratio of 1.2 is needed for efficient magnetic cell shepherding, whereas enhanced MFH-induced mortality (70 vs. 15%) can be reached with hybrids enriched in the coercive Fe3C phase. These results suggest that a synergistic effect between the Ab loading and the Fe distribution in each nanotube exists, for which the maximum shepherding and hyperthermia effects are observed when higher densities of Fe@CNTs featuring the more coercive phase are interfaced with the cells.Fe-filled carbon nanotubes (Fe@CNTs) recently emerged as an effective class of hybrid nanoparticles for biotechnological applications, such as magnetic cell sorting and magnetic fluid hyperthermia. Aiming at studying the effects of both the Fe loading and the magnetocrystalline characteristics in these applications, we describe herein the preparation of Fe@CNTs containing different Fe phases that, upon functionalization with the antibody Cetuximab (Ctxb), allow the targeting of cancer cells. Our experimental findings reveal that an optimal Ctxb/Fe weight ratio of 1.2 is needed for efficient magnetic cell shepherding, whereas enhanced MFH-induced mortality (70 vs. 15%) can be reached with hybrids enriched in the coercive Fe3C phase. These results suggest that a synergistic effect between the Ab loading and the Fe distribution in each nanotube exists, for which the maximum shepherding and hyperthermia effects are observed when higher densities of Fe

  4. Investigating the fluid mechanics behind red blood cell-induced lateral platelet motion

    NASA Astrophysics Data System (ADS)

    Crowl Erickson, Lindsay; Fogelson, Aaron

    2009-11-01

    Platelets play an essential role in blood clotting; they adhere to damaged tissue and release chemicals that activate other platelets. Yet in order to adhere, platelets must first come into contact with the injured vessel wall. Under arterial flow conditions, platelets have an enhanced concentration near blood vessel walls. This non-uniform cell distribution depends on the fluid dynamics of blood as a heterogeneous medium. We use a parallelized lattice Boltzmann-immersed boundary method to solve the flow dynamics of red cells and platelets in a periodic 2D vessel with no-slip boundary conditions. Red cells are treated as biconcave immersed boundary objects with isotropic Skalak membrane tension and an internal viscosity five times that of the surrounding plasma. Using this method we analyze the influence of shear rate, hematocrit, and red cell membrane properties on lateral platelet motion. We find that the effective diffusion of platelets is significantly lower near the vessel wall compared to the center of the vessel. Insight gained from this work could lead to significant improvements to current models for platelet adhesion where the presence of red blood cells is neglected due to computational intensity.

  5. Design of an ultrashort optical transmission cell for vacuum ultraviolet spectroscopy of supercritical fluids.

    PubMed

    Janik, Ireneusz; Marin, Timothy W

    2015-01-01

    We present the design and characteristics of an ultrathin flow cell optimized for vacuum ultraviolet transmission spectroscopy experiments on supercritical fluids. The cell operates satisfactorily at pressures up to 300 bar and temperatures up to 390 °C. The variable path length concept of the cell allows for optical transmission studies of analytes ranging from dense condensed-phase systems to gas-phase systems. The path length of the cell can be adjusted from hundreds of nanometers to hundreds of micrometers by an exchange of a variable thickness spacer sandwiched between two sapphire windows. In the path length range from nanometers to single micrometers, metal vapor deposited on one or both of the two sandwiched optical windows constitute the spacer. Spacers with thicknesses of 2 μm and greater can be constructed from simple commercially available metal foils. The cell has been used to measure the lowest-lying absorption band of water in both the vapor and condensed phases from room temperature up to and above the critical point. It has also found application in the studies of aqueous ions and nonaqueous liquids including various common organic solvents and carbon dioxide. PMID:25638117

  6. Three-Dimensional Computational Fluid Dynamics Modeling of Solid Oxide Electrolysis Cells and Stacks

    SciTech Connect

    Grant Hawkes; James O'Brien; Carl Stoots; Stephen Herring

    2008-07-01

    A three-dimensional computational fluid dynamics (CFD) electrochemical model has been created for detailed analysis of a high-temperature electrolysis stack (solid oxide fuel cells operated as electrolyzers). Inlet and outlet plenum flow distributions are discussed. Maldistribution of plena flow show deviations in per-cell operating conditions due to non-uniformity of species concentrations. Models have also been created to simulate experimental conditions and for code validation. Comparisons between model predictions and experimental results are discussed. Mass, momentum, energy, and species conservation and transport are provided via the core features of the commercial CFD code FLUENT. A solid-oxide fuel cell (SOFC) model adds the electrochemical reactions and loss mechanisms and computation of the electric field throughout the cell. The FLUENT SOFC user-defined subroutine was modified for this work to allow for operation in the electrolysis mode. Model results provide detailed profiles of temperature, Nernst potential, operating potential, activation over-potential, anode-side gas composition, cathode-side gas composition, current density and hydrogen production over a range of stack operating conditions. Variations in flow distribution, and species concentration are discussed. End effects of flow and per-cell voltage are also considered. Predicted mean outlet hydrogen and steam concentrations vary linearly with current density, as expected. Contour plots of local electrolyte temperature, current density, and Nernst potential indicate the effects of heat transfer, reaction cooling/heating, and change in local gas composition.

  7. The use of human amniotic fluid mesenchymal stem cells as the feeder layer to establish human embryonic stem cell lines.

    PubMed

    Soong, Yung-Kwei; Huang, Shang-Yu; Yeh, Chiu-Hsiang; Wang, Tzu-Hao; Chang, Kuo-Hsuan; Cheng, Po-Jen; Shaw, S W Steven

    2015-12-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have the potential to differentiate into the three germ layers and possibly all tissues of the human body. To fulfil the clinical potentials for cell-based therapy, banks of hESC lines that express different combinations of the major histocompatibility genes should be established, preferably without exposing such cells to animal cells and proteins. In this study, we tested human amniotic fluid mesenchymal stem cells (AFMSCs) as feeder cells to support the growth of hESCs. Our results indicated that mitomycin-treated AFMSCs were able to support the newly established hESC lines CGLK-1 and CGLK-2. The hESC colonies cultured on AFMSCs expressed alkaline phosphatase (ALK-P), SSEA-4, TRA-1-60, TRA-1-81, Oct-4, Nanog and Sox-2, which are markers for undifferentiated hESCs. Chromosomal analyses of both hESC lines, CGLK-1 and CGLK-2, which were cultured on AFMSC feeders for 22 and 14 passages, respectively, were confirmed to be normal karyotypes (46, XX). The ability of AFMSCs as feeder cells to maintain the undifferentiated growth and pluripotency of hESCs was confirmed by in vivo formation of teratomas derived on AFMSC hESCs in severe combined immune-compromised mice. The use of AFMSCs for feeder cells to culture hESCs has several advantages, in that AFMSCs are not tumourigenic and can be expanded extensively with a short doubling time. PMID:23460275

  8. Tapping and contact mode imaging of native chromosomes and extraction of genomic DNA using AFM tips

    NASA Astrophysics Data System (ADS)

    Sun, Yingchun; Arakawa, Hideo; Osada, Toshiya; Ikai, Atsushi

    2002-03-01

    It is very important both in medicine and biology to clarify the chromosomal structure to understand its functions. In a standard cytogenetic procedure, chromosomes are often fixed in a mixture of acetic acid and methanol. This process most likely changes the mechanical property of chromosomes. We adopted a method to prepare native and unfixed chromosomes from mouse 3T3 cells and used tapping and contact mode atomic force microscopy (AFM) to image and manipulate them. Modified AFM tips were used to image chromosomes in contact mode in air, and then the chromosome samples were immobilized on a substrate and placed in a buffer solution to pull out DNA-histone complexes from them after they were optimally treated with trypsin. From the AFM images, we could see several bands and granular structures on chromosomes. We obtained force curves indicating long fiber extensions from native chromosomes both with low (in high concentration of NaCl) and high forces (physiological conditions). The result suggested that the degree of chromosome condensation decreased in high concentration of salt. It agrees with the known fact of histone H1 dissociation in a high concentration of salt. We intend to pull out DNA-histone complexes from chromosomes for later molecular operations on them using an AFM.

  9. Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis

    PubMed Central

    Savickiene, Jurate; Treigyte, Grazina; Baronaite, Sandra; Valiuliene, Giedre; Kaupinis, Algirdas; Valius, Mindaugas; Arlauskiene, Audrone; Navakauskiene, Ruta

    2015-01-01

    Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications. PMID:26351462

  10. Fluid-Structure Interactions Analysis of Shear-Induced Modulation of a Mesenchymal Stem Cell: An Image-Based Study.

    PubMed

    Ghaemi, Roza Vaez; Vahidi, Bahman; Sabour, Mohammad Hossein; Haghighipour, Nooshin; Alihemmati, Zakieh

    2016-03-01

    Although effects of biochemical modulation of stem cells have been widely investigated, only recent advances have been made in the identification of mechanical conditioning on cell signaling pathways. Experimental investigations quantifying the micromechanical environment of mesenchymal stem cells (MSCs) are challenging while computational approaches can predict their behavior due to in vitro stimulations. This study introduces a 3D cell-specific finite element model simulating large deformations of MSCs. Here emphasizing cell mechanical modulation which represents the most challenging multiphysics phenomena in sub-cellular level, we focused on an approach attempting to elicit unique responses of a cell under fluid flow. Fluorescent staining of MSCs was performed in order to visualize the MSC morphology and develop a geometrically accurate model of it based on a confocal 3D image. We developed a 3D model of a cell fixed in a microchannel under fluid flow and then solved the numerical model by fluid-structure interactions method. By imposing flow characteristics representative of vigorous in vitro conditions, the model predicts that the employed external flow induces significant localized effective stress in the nucleo-cytoplasmic interface and average cell deformation of about 40%. Moreover, it can be concluded that a lower strain level is made in the cell by the oscillatory flow as compared with steady flow, while same ranges of effective stress are recorded inside the cell in both conditions. The deeper understanding provided by this study is beneficial for better design of single cell in vitro studies. PMID:26333040

  11. Use of simulated intestinal fluids with Caco-2 cells and rat ileum.

    PubMed

    Patel, N; Forbes, B; Eskola, S; Murray, J

    2006-02-01

    In most in vitro studies of oral drug permeability, little attempt is made to reproduce the gastrointestinal lumenal environment. The aim of this study was to evaluate the compatibility of simulated intestinal fluid (SIF) solutions with Caco-2 cell monolayers and Ussing chamber-mounted rat ileum under standard permeability experiment protocols. In preliminary experiments, fasted-state simulated intestinal fluid (FaSSIF) and fed-state simulated intestinal fluid (FeSSIF) solutions based on the dissolution medium formulae of Dressman and co-workers (1998) were modified for compatibility with Caco-2 cells to produce FaS-SIF and FeSSIF "transport" solutions for use with in vitro permeability models. For Caco-2 cells exposed to FaSSIF and FESSIF transport solutions, the transepithelial electrical resistance was maintained for over 4 h and mannitol permeability was equivalent to that in control (Hank's Balanced Salt Solution-treated) cell layers. Scanning electron microscopy revealed that microvilli generally maintained a normal distribution, although some shortening of microvilli and occasional small areas of denudation were observed. For rat ileum in the Ussing chambers, the potential difference (PD) collapsed to zero over 120 min when exposed to the FaSSIF transport solution and an even faster collapse of the PD was observed when the FeSSIF transport solution was used. Electron micrographs revealed erosion of the villi tips and substantial denudation of the microvilli after exposure of ileal tissue to FaSSIF and FeSSIF solutions, and permeability to mannitol was increased by almost two-fold. This study indicated that FaSSIF and FeSSIF transport solutions can be used with Caco-2 monolayers to evaluate drug permeability, but rat ileum in Ussing chambers is adversely affected by these solutions. Metoprolol permeability in Caco-2 experiments was reduced by 33% using the FaSSIF and 75% using the FeSSIF compared to permeability measured using HBSS. This illustrates that

  12. Segmental calibration for commercial AFM in vertical direction

    NASA Astrophysics Data System (ADS)

    Shi, Yushu; Gao, Sitian; Lu, Mingzhen; Li, Wei; Xu, Xuefang

    2013-01-01

    Atomic force microscopy (AFM) is most widely applied in scientific research and industrial production. AFM is a scanning probe imaging and measuring device, useful for physical and chemical studies. Depends on its basic structure, microscopic surface pattern can be measured and captured by mechanically scanning. Its vertical and horizon resolution can reach to 0.01nm and 0.1nm. Commonly the measurement values of commercial AFM are directly from scanning piezoelectric tube, so that it not a traceable value. In order to solve the problem of commercial AFM's traceability, step height standard references are applied to calibrate the piezoelectric ceramic housing in scanning tube. All of the serial of step height standard references, covering the commercial AFM vertical scale, are calibrated by Metrology AFM developed by National Institute of Metrology (NIM), China. Three interferometers have been assembled in its XYZ axis, therefore the measurement value can directly trace to laser wavelength. Because of nonlinear characteristic of PZT, the method of segmental calibration is proposed. The measurement scale can be divided into several subsections corresponding to the calibrated values of the series of step height standards references. By this method the accuracy of measurements can be ensured in each segment measurement scale and the calibration level of the whole instrument can be promoted. In order to get a standard step shape by commercial AFM, substrate removal method is applied to deal with the bow shape problem.

  13. Spatiotemporal properties of intracellular calcium signaling in osteocytic and osteoblastic cell networks under fluid flow.

    PubMed

    Jing, Da; Lu, X Lucas; Luo, Erping; Sajda, Paul; Leong, Pui L; Guo, X Edward

    2013-04-01

    Mechanical stimuli can trigger intracellular calcium (Ca(2+)) responses in osteocytes and osteoblasts. Successful construction of bone cell networks necessitates more elaborate and systematic analysis for the spatiotemporal properties of Ca(2+) signaling in the networks. In the present study, an unsupervised algorithm based on independent component analysis (ICA) was employed to extract the Ca(2+) signals of bone cells in the network. We demonstrated that the ICA-based technology could yield higher signal fidelity than the manual region of interest (ROI) method. Second, the spatiotemporal properties of Ca(2+) signaling in osteocyte-like MLO-Y4 and osteoblast-like MC3T3-E1 cell networks under laminar and steady fluid flow stimulation were systematically analyzed and compared. MLO-Y4 cells exhibited much more active Ca(2+) transients than MC3T3-E1 cells, evidenced by more Ca(2+) peaks, less time to the 1st peak and less time between the 1st and 2nd peaks. With respect to temporal properties, MLO-Y4 cells demonstrated higher spike rate and Ca(2+) oscillating frequency. The spatial intercellular synchronous activities of Ca(2+) signaling in MLO-Y4 cell networks were higher than those in MC3T3-E1 cell networks and also negatively correlated with the intercellular distance, revealing faster Ca(2+) wave propagation in MLO-Y4 cell networks. Our findings show that the unsupervised ICA-based technique results in more sensitive and quantitative signal extraction than traditional ROI analysis, with the potential to be widely employed in Ca(2+) signaling extraction in the cell networks. The present study also revealed a dramatic spatiotemporal difference in Ca(2+) signaling for osteocytic and osteoblastic cell networks in processing the mechanical stimulus. The higher intracellular Ca(2+) oscillatory behaviors and intercellular coordination of MLO-Y4 cells provided further evidences that osteocytes may behave as the major mechanical sensor in bone modeling and remodeling

  14. Phosphodiesterase Isoform Regulation of Cell Proliferation and Fluid Secretion in Autosomal Dominant Polycystic Kidney Disease.

    PubMed

    Pinto, Cibele S; Raman, Archana; Reif, Gail A; Magenheimer, Brenda S; White, Corey; Calvet, James P; Wallace, Darren P

    2016-04-01

    cAMP stimulates cell proliferation and Cl(-)-dependent fluid secretion, promoting the progressive enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). Intracellular cAMP levels are determined by the balance of cAMP synthesis by adenylyl cyclases and degradation by phosphodiesterases (PDEs). Therefore, PDE isoform expression and activity strongly influence global and compartmentalized cAMP levels. We report here that PDE3 and PDE4 expression levels are lower in human ADPKD tissue and cells compared with those of normal human kidneys (NHKs), whereas PDE1 levels are not significantly different. Inhibition of PDE4 caused a greater increase in basal and vasopressin (AVP)-stimulated cAMP levels and Cl(-) secretion by ADPKD cells than inhibition of PDE1, and inhibition of PDE4 induced cyst-like dilations in cultured mouse Pkd1(-/-) embryonic kidneys. In contrast, inhibition of PDE1 caused greater stimulation of extracellular signal-regulated kinase (ERK) and proliferation of ADPKD cells than inhibition of PDE4, and inhibition of PDE1 enhanced AVP-induced ERK activation. Notably, inhibition of PDE1, the only family of Ca(2+)-regulated PDEs, also induced a mitogenic response to AVP in NHK cells, similar to the effect of restricting intracellular Ca(2+). PDE1 coimmunoprecipitated with B-Raf and A-kinase anchoring protein 79, and AVP increased this interaction in ADPKD but not NHK cells. These data suggest that whereas PDE4 is the major PDE isoform involved in the regulation of global intracellular cAMP and Cl(-) secretion, PDE1 specifically affects the cAMP signal to the B-Raf/MEK/ERK pathway and regulates AVP-induced proliferation of ADPKD cells. PMID:26289612

  15. Tethered cells in fluid flows--beyond the Stokes' drag force approach.

    PubMed

    Zakrisson, Johan; Wiklund, Krister; Axner, Ove; Andersson, Magnus

    2015-10-01

    Simulations of tethered cells in viscous sub-layers are frequently performed using the Stokes' drag force, but without taking into account contributions from surface corrections, lift forces, buoyancy, the Basset force, the cells' finite inertia, or added mass. In this work, we investigate to what extent such contributions, under a variety of hydrodynamic conditions, influence the force at the anchor point of a tethered cell and the survival probability of a bacterium that is attached to a host by either a slip or a catch bond via a tether with a few different biomechanical properties. We show that a consequence of not including some of these contributions is that the force to which a bond is exposed can be significantly underestimated; in general by ∼32-46%, where the influence of the surface corrections dominate (the parallel and normal correction coefficients contribute ∼5-8 or ∼23-26%, respectively). The Basset force is a major contributor, up to 20%, for larger cells and shear rates. The lift force and inertia contribute when cells with radii >3 μm have shear rates >2000 s(-1). Buoyancy contributes significantly for cells with radii >3 μm for shear rates <10 s(-1). Since the lifetime of a bond depends strongly on the force, both the level of approximation and the biomechanical model of the tether significantly affect the survival probability of tethered bacteria. For a cell attached by a FimH-mannose bond and an extendable tether with a shear rate of 3000 s(-1), neglecting the surface correction coefficients or the Basset force can imply that the survival probability is overestimated by more than an order of magnitude. This work thus shows that in order to quantitatively assess bacterial attachment forces and survival probabilities, both the fluid forces and the tether properties need to be modeled accurately. PMID:26331992

  16. Differences in cerebrospinal fluid inflammatory cell reaction of patients with leptomeningeal involvement by lymphoma and carcinoma.

    PubMed

    Illán, Julia; Simo, Marta; Serrano, Cristina; Castañón, Susana; Gonzalo, Raquel; Martínez-García, María; Pardo, Javier; Gómez, Lidia; Navarro, Miguel; Altozano, Javier Pérez; Alvarez, Ruth; Bruna, Jordi; Subirá, Dolores

    2014-12-01

    Dissemination of neoplastic cells into the cerebrospinal fluid (CSF) and leptomeninges is a devastating complication in patients with epithelial cell neoplasia (leptomeningeal carcinomatosis [LC]) and lymphomas (lymphomatous meningitis [LyM]). Information about the surrounding inflammatory cell populations is scarce. In this study, flow cytometry immunophenotyping was used to describe the distribution of the main leukocyte populations in the CSF of 83 patients diagnosed with neoplastic meningitis (LC, n = 65; LyM, n = 18). These data were compared with those obtained in the CSF from 55 patients diagnosed with the same groups of neoplasia without meningeal involvement (solid tumors, n = 36; high-grade lymphoma, n = 19). Median (interquartile) rates of lymphocytes, monocytes, and polymorphonuclear (PMN) cells were 59.7% (range, 35-76.6%), 24% (range, 16-53%), and 1.5% (range, 0-7.6%) in LC, respectively, and 98.5% (range, 70.8-100%), 1.5% (range, 0-29.3%), and 0% in LyM, respectively (P < 0.001). No difference was observed between patients with breast adenocarcinoma (n = 30) and lung adenocarcinoma (n = 21), nor with different rates of malignant CSF involvement. Patients with lymphoma (with or without LyM) had a similar CSF leukocyte distribution, but cancer patients with LC and without LC had a distinctive PMN cell rate (P = 0.002). These data show that CSF samples from patients with LC have a greater number of inflammatory cells and a different leukocyte distribution than seen in the CSF from patients with LyM. Description of PMN cells is a distinctive parameter of patients with LC, compared with the CSF from patients with LyM and patients with cancer but without LC. PMID:24746871

  17. A study of water droplet between an AFM tip and a substrate using dissipative particle dynamics

    NASA Astrophysics Data System (ADS)

    Pal, Souvik; Lan, Chuanjin; Li, Zhen; Hirleman, E. Daniel; Ma, Yanbao

    2014-11-01

    Formation of a water droplet between a sharp AFM tip and a substrate due to capillary condensation affects the tip-substrate interaction. As a consequence, AFM measurements lose precision and often produce incorrect sample topology. Understanding the physics of liquid bridges is also important in the field of Dip-pen nanolithography (DPN). Significant research is being carried out to understand the mechanics of the formation of the liquid bridge and its dependence of surface properties, ambient conditions etc. The in-between length scale, i.e., mesoscale (~100 nm) associated with this phenomenon presents a steep challenge for experimental measurements. In addition, molecular dynamics (MD) can be computationally prohibitive to model the entire system, especially over microseconds to seconds. Theoretical analysis using Young Laplace equation has so far provided some qualitative insights only. We study this system using Dissipative Particle Dynamics (DPD) which is a simulation technique suitable for describing mesoscopic hydrodynamic behavior of fluids. In this work, we carry out simulations to improve understanding of the process of formation of the meniscus, the mechanics of manipulation and control of its shape, and better estimation of capillary forces. The knowledge gained through our study will help in correcting the AFM measurements affected by capillary condensation. Moreover, it will improve understanding of more accurate droplet manipulation in DPN.

  18. Reflex Testing Rules for Cell Count and Differentiation of Nucleated Elements in Pleural and Ascitic Fluids on Sysmex XE-5000.

    PubMed

    Buoro, Sabrina; Appassiti Esposito, Sara; Vavassori, Mauro; Mecca, Tommaso; Ottomano, Cosimo; Dominoni, Paola; Seghezzi, Michela; Candiago, Elisabetta; Farina, Claudio; Gianatti, Andrea; Crippa, Alberto; Lippi, Giuseppe

    2016-04-01

    Flow cytometry is widely used in many laboratories for automated nucleated cell counts and their differentiation in body fluids. The implementation of new reflex testing rules on these automated instruments could open new frontiers in laboratory workflow, improving characterization of body fluids and clinical diagnosis and decreasing costs. Ascitic (150) and pleural (33) fluids were collected and assessed by XE-5000 and optical microscopy. Cell counts performed with the methods showed a Pearson's correlation of 0.98 (p < 0.0001), Passing-Bablok regression y = 0.99x + 2.44, and bias of 32.3. In ascitic fluids, the best diagnostic performance was found for polymorphonuclear and neutrophil counts on XE-5000, which exhibited areas under the curve (AUCs) 0.98 (p < 0.0001) and 0.99 (p < 0.0001), respectively. In pleural fluids the best diagnostic performance was found for polymorphonuclear percent parameter, which displayed 0.97 (p < 0.0001). Specific reflex test rules based on these parameters were characterized by 92% diagnostic concordance, 1.00 sensitivity, and 0.84 specificity with optical microscopy. The application of a set of reflex testing rules may improve the diagnostic performance of XE-5000, increasing its reliability for routine automated cell count in body fluids. We acknowledge that further studies should be planned to validate our findings according to clinical data. PMID:26149816

  19. Fluid shear promotes chondrosarcoma cell invasion by activating matrix metalloproteinase 12 via IGF-2 and VEGF signaling pathways.

    PubMed

    Wang, P; Chen, S-H; Hung, W-C; Paul, C; Zhu, F; Guan, P-P; Huso, D L; Kontrogianni-Konstantopoulos, A; Konstantopoulos, K

    2015-08-27

    Interstitial fluid flow in and around the tumor tissue is a physiologically relevant mechanical signal that regulates intracellular signaling pathways throughout the tumor. Yet, the effects of interstitial flow and associated fluid shear stress on the tumor cell function have been largely overlooked. Using in vitro bioengineering models in conjunction with molecular cell biology tools, we found that fluid shear (2 dyn/cm(2)) markedly upregulates matrix metalloproteinase 12 (MMP-12) expression and its activity in human chondrosarcoma cells. MMP-12 expression is induced in human chondrocytes during malignant transformation. However, the signaling pathway regulating MMP-12 expression and its potential role in human chondrosarcoma cell invasion and metastasis have yet to be delineated. We discovered that fluid shear stress induces the synthesis of insulin growth factor-2 (IGF-2) and vascular endothelial growth factor (VEGF) B and D, which in turn transactivate MMP-12 via PI3-K, p38 and JNK signaling pathways. IGF-2-, VEGF-B- or VEGF-D-stimulated chondrosarcoma cells display markedly higher migratory and invasive potentials in vitro, which are blocked by inhibiting MMP-12, PI3-K, p38 or JNK activity. Moreover, recombinant human MMP-12 or MMP-12 overexpression can potentiate chondrosarcoma cell invasion in vitro and the lung colonization in vivo. By reconstructing and delineating the signaling pathway regulating MMP-12 activation, potential therapeutic strategies that interfere with chondrosarcoma cell invasion may be identified. PMID:25435370

  20. AMPK agonists ameliorate sodium and fluid transport and inflammation in cystic fibrosis airway epithelial cells.

    PubMed

    Myerburg, Michael M; King, J Darwin; Oyster, Nicholas M; Fitch, Adam C; Magill, Amy; Baty, Catherine J; Watkins, Simon C; Kolls, Jay K; Pilewski, Joseph M; Hallows, Kenneth R

    2010-06-01

    The metabolic sensor AMP-activated kinase (AMPK) inhibits both the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel and epithelial Na(+) channel (ENaC), and may inhibit secretion of proinflammatory cytokines in epithelia. Here we have tested in primary polarized CF and non-CF human bronchial epithelial (HBE) cells the effects of AMPK activators, metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR), on various parameters that contribute to CF lung disease: ENaC-dependent short-circuit currents (I(sc)), airway surface liquid (ASL) height, and proinflammatory cytokine secretion. AMPK activation after overnight treatment with either metformin (2-5 mM) or AICAR (1 mM) substantially inhibited ENaC-dependent I(sc) in both CF and non-CF airway cultures. Live-cell confocal images acquired 60 minutes after apical addition of Texas Red-dextran-containing fluid revealed significantly greater ASL heights after AICAR and metformin treatment relative to controls, suggesting that AMPK-dependent ENaC inhibition slows apical fluid reabsorption. Both metformin and AICAR decreased secretion of various proinflammatory cytokines, both with and without prior LPS stimulation. Finally, prolonged exposure to more physiologically relevant concentrations of metformin (0.03-1 mM) inhibited ENaC currents and decreased proinflammatory cytokine levels in CF HBE cells in a dose-dependent manner. These findings suggest that novel therapies to activate AMPK in the CF airway may be beneficial by blunting excessive sodium and ASL absorption and by reducing excessive airway inflammation, which are major contributors to CF lung disease. PMID:19617399

  1. Raman and AFM study of gamma irradiated plastic bottle sheets

    SciTech Connect

    Ali, Yasir; Kumar, Vijay; Dhaliwal, A. S.; Sonkawade, R. G.

    2013-02-05

    In this investigation, the effects of gamma irradiation on the structural properties of plastic bottle sheet are studied. The Plastic sheets were exposed with 1.25MeV {sup 60}Co gamma rays source at various dose levels within the range from 0-670 kGy. The induced modifications were followed by micro-Raman and atomic force microscopy (AFM). The Raman spectrum shows the decrease in Raman intensity and formation of unsaturated bonds with an increase in the gamma dose. AFM image displays rough surface morphology after irradiation. The detailed Raman analysis of plastic bottle sheets is presented here, and the results are correlated with the AFM observations.

  2. Dynamics of the nanoneedle probe in trolling mode AFM.

    PubMed

    Abdi, Ahmad; Pishkenari, Hossein Nejat; Keramati, Ramtin; Minary-Jolandan, Majid

    2015-05-22

    Atomic force microscopy (AFM), as an indispensable tool for nanoscale characterization, presents major drawbacks for operation in a liquid environment arising from the large hydrodynamic drag on the vibrating cantilever. The newly introduced 'Trolling mode' (TR-mode) AFM resolves this complication by using a specialized nanoneedle cantilever that keeps the cantilever outside of the liquid. Herein, a mechanical model with a lumped mass was developed to capture the dynamics of such a cantilever with a nanoneedle tip. This new developed model was applied to investigate the effects of the needle-liquid interface on the performance of the AFM, including the imaging capability in liquid. PMID:25915451

  3. AFM Studies of Conformational Changes in Proteins and Peptides

    NASA Astrophysics Data System (ADS)

    Ploscariu, Nicoleta; Sukthankar, Pinakin; Tomich, John; Szoszkiewicz, Robert

    2015-03-01

    Here, we present estimates of molecular stiffness and mechanical energy dissipation factors for some examples of proteins and peptides. The results are obtained from AFM force spectroscopy measurements. To determine molecular stiffness and mechanical energy dissipation factors we developed a model based on measuring several resonance frequencies of an AFM cantilever in contact with either single protein molecule or peptides adsorbed on arbitrary surface. We used compliant AFM cantilevers with a small aspect ratio - a ratio of length to width - in air and in liquid, including biologically relevant phosphate buffered saline medium. Department of Physics.

  4. Ethanol alters the osteogenic differentiation of amniotic fluid-derived stem cells

    PubMed Central

    Hipp, Jennifer A; Hipp, Jason D; Atala, Anthony; Soker, Shay

    2010-01-01

    Background Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental defects caused by prenatal alcohol exposure. Clinical manifestations of FASD are highly variable and include mental retardation and developmental defects of the heart, kidney, muscle, skeleton, and craniofacial structures. Specific effects of ethanol on fetal cells include induction of apoptosis as well as inhibition of proliferation, differentiation, and migration. This complex set of responses suggests that a bioinformatics approach could clarify some of the pathways involved in these responses. Methods In this study, the responses of fetal stem cells derived from the amniotic fluid (AFSCs) to treatment with ethanol have been examined. Large-scale transcriptome analysis of ethanol-treated AFSCs indicates that genes involved in skeletal development and ossification are up-regulated in these cells. Therefore, the effect of ethanol on osteogenic differentiation of AFSCs was studied. Results Exposure to ethanol during the first 48 hours of an osteogenic differentiation protocol increased in vitro calcium deposition by AFSCs and increased alkaline phosphatase activity. In contrast, ethanol treatment later in the differentiation protocol (day 8) had no significant effect on the activity of alkaline phosphatase. Conclusions These results suggest that transient exposure of AFSCs to ethanol during early differentiation enhances osteogenic differentiation of the cells. PMID:20608908

  5. Nanoscopic polypyrrole AFM-SECM probes enabling force measurements under potential control

    NASA Astrophysics Data System (ADS)

    Knittel, P.; Higgins, M. J.; Kranz, C.

    2014-01-01

    Conductive polymers, and in particular polypyrrole, are frequently used as biomimetic interfaces facilitating growth and/or differentiation of cells and tissues. Hence, studying forces and local interactions between such polymer interfaces and cells at the nanoscale is of particular interest. Frequently, such force interactions are not directly accessible with high spatial resolution. Consequently, we have developed nanoscopic polypyrrole electrodes, which are integrated in AFM-SECM probes. Bifunctional AFM-SECM probes were modified via ion beam-induced deposition resulting in pyramidal conductive Pt-C composite electrodes. These nanoscopic electrodes then enabled localized polypyrrole deposition, thus resulting in polymer-modified AFM probes with a well-defined geometry. Furthermore, such probes may be reversibly switched from an insulating to a conductive state. In addition, the hydrophilicity of such polymer tips is dependent on the dopant, and hence, on the oxidation state. Force studies applying different tip potentials were performed at plasma-treated glass surfaces providing localized information on the associated force interactions, which are dependent on the applied potential and the dopant.Conductive polymers, and in particular polypyrrole, are frequently used as biomimetic interfaces facilitating growth and/or differentiation of cells and tissues. Hence, studying forces and local interactions between such polymer interfaces and cells at the nanoscale is of particular interest. Frequently, such force interactions are not directly accessible with high spatial resolution. Consequently, we have developed nanoscopic polypyrrole electrodes, which are integrated in AFM-SECM probes. Bifunctional AFM-SECM probes were modified via ion beam-induced deposition resulting in pyramidal conductive Pt-C composite electrodes. These nanoscopic electrodes then enabled localized polypyrrole deposition, thus resulting in polymer-modified AFM probes with a well

  6. A quick method for concentrating and processing cancer cells from serous fluids and fine-needle nodule aspirates.

    PubMed

    Elequin, F T; Muggia, F M; Ghossein, N A; Schreiber, K

    1977-01-01

    A quick method of concentrating cancer cells and the preparation of cytological slides from body fluids and aspirates is described, using a single Ficoll gradient and cytocentrifuge. The method eliminates the disadvantages of conventional techniques, i.e., excess contamination by red (erythrocytes) or white (leukocytes) blood cells and a scarcity of cancer cells. Because the technique is simple and requires only standard cytotechnological equipment, it can be easily adopted as an aid to diagnostic routine in cancer cytology. PMID:269612

  7. The value of multiparameter flow cytometry of cerebrospinal fluid involved by leukemia/lymphoma cells.

    PubMed

    Babusíková, O; Zelezníková, T

    2004-01-01

    The usefulness of multiparameter flow cytometric (FC) analysis of cerebrospinal fluid (CSF) was evaluated in leukemia/lymphoma patients having central nervous system (CNS) involvement of the disease. In 12 specimens of 8 patients with different types of leukemia/lymphoma (one case of T-ALL, 3 cases of early B-cell ALL, one case of AML, and 3 proven or suspicious NHL cases) the presence of pathological clone in CSF has been confirmed or excluded. The phenotypic patterns of CSF cells were defined according to those of bone marrow (BM)/peripheral blood (PB) at diagnosis or during follow-up of the same patients. Furthermore, in one case of suspicious CNS infiltration of NHL, the pathological clone was characterized as a highly suspicious of solid tumor and was proved to be a lung cancer metastasis. The definition was made on the basis of CD45 (common leukocyte antigen) and other studied CD markers negativity. The exact comparison of immunophenotypic profiles of specimens from different sites (CSF, BM, PB) of the same patient has been performed and no phenotypic changes were found. In some CSF specimens, where no cells of suspicious pathological clone were detected, in 4-color analysis only normal lymphocyte population was found even in small cell samples (even if the cellularity was < than 0.3x10-6). In these populations the high values of T-cells (CD3+) predominated and the high prevalence of CD4+ over CD8+ cells, and an almost total lack of B-lymphocytes was found. Our results suggest that positive CSF immunology is a useful indicator of malignancy and reflects leptomeningeal involvement. Simultaneously we demonstrated that FC analysis of CSF in the aim to detect possible CSF seeding of leukemia/lymphoma is a reliable and quick technique. PMID:15640938

  8. Tethered cells in fluid flows—beyond the Stokes’ drag force approach

    NASA Astrophysics Data System (ADS)

    Zakrisson, Johan; Wiklund, Krister; Axner, Ove; Andersson, Magnus

    2015-10-01

    Simulations of tethered cells in viscous sub-layers are frequently performed using the Stokes’ drag force, but without taking into account contributions from surface corrections, lift forces, buoyancy, the Basset force, the cells’ finite inertia, or added mass. In this work, we investigate to what extent such contributions, under a variety of hydrodynamic conditions, influence the force at the anchor point of a tethered cell and the survival probability of a bacterium that is attached to a host by either a slip or a catch bond via a tether with a few different biomechanical properties. We show that a consequence of not including some of these contributions is that the force to which a bond is exposed can be significantly underestimated; in general by ˜32-46%, where the influence of the surface corrections dominate (the parallel and normal correction coefficients contribute ˜5-8 or ˜23-26%, respectively). The Basset force is a major contributor, up to 20%, for larger cells and shear rates. The lift force and inertia contribute when cells with radii >3 μm have shear rates >2000 s-1. Buoyancy contributes significantly for cells with radii >3 μm for shear rates <10 s-1. Since the lifetime of a bond depends strongly on the force, both the level of approximation and the biomechanical model of the tether significantly affect the survival probability of tethered bacteria. For a cell attached by a FimH-mannose bond and an extendable tether with a shear rate of 3000 s-1, neglecting the surface correction coefficients or the Basset force can imply that the survival probability is overestimated by more than an order of magnitude. This work thus shows that in order to quantitatively assess bacterial attachment forces and survival probabilities, both the fluid forces and the tether properties need to be modeled accurately.

  9. Vibration Induced Osteogenic Commitment of Mesenchymal Stem Cells is Enhanced by Cytoskeletal Remodeling but not Fluid Shear

    PubMed Central

    Uzer, Gunes; Pongkitwitoon, Suphannee; Chan, M Ete; Judex, Stefan

    2013-01-01

    Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell’s sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04Pa to 5Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100Hz and 30Hz. After 14d and under low fluid shear conditions associated with 100Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30Hz vibrations enhanced mineralization only in the 2g group. Over 3d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott-Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity. PMID:23870506

  10. Study of cell apoptosis in the hippocampus and thalamencephalon in a ventricular fluid impact model

    PubMed Central

    CHEN, RUI; WANG, JUNYU; JIANG, BING; WAN, XIN; LIU, HONGWEI; LIU, HUAN; YANG, XIAOSHENG; WU, XIAOBING; ZOU, QIN; YANG, WENREN

    2013-01-01

    The aim of this study was to investigate the apoptosis of nerve cells in the hippocampal and thalamencephalon regions using a rabbit model of ventricular fluid impact. The results for the study demonstrated a variety of pathophysiological changes in the rabbit model, while changes in the hippocampal and thalamencephalon regions were observed under a light microscope following hematoxylin and eosin (H&E)/terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Among the mild, moderate and severe injury groups, there were significant differences in the mortality rate and in the changes in vital signs and consciousness recovery time following trauma. Furthermore, H&E staining showed that pathological changes, such as hemorrhage and necrosis, occurred in the hippocampal and thalamencephalon regions at an early stage subsequent to trauma, while TUNEL staining showed that neuronal apoptosis occurred in the various injury groups. In traumatic brain injuries, the impact caused by cerebrospinal fluid moving with a certain energy results in marked damage to the contralateral periventricular structures and may generate a series of pathophysiological changes. PMID:24255676

  11. Instability of displacement of Oldroyd-B fluid by air in a Hele-Shaw cell

    NASA Astrophysics Data System (ADS)

    Daripa, Prabir

    2014-03-01

    We study the displacement of an Oldroyd-B fluid in a Hele-Shaw cell when driven by air. In particular, we explicitly obtain an analytical expression for the growth rate of instability which depends on the relaxation and retardation (time) constants, denoted by λ, and λ1 respectively, appearing in the Oldroyd-B constitutive relations. When these two constants are zero, we recover the classical Saffman-Taylor result for a Newtonian liquid displaced by air. Our results show that this displacement process is more unstable than the case when a Newtonian fluid is displaced by air. The analytical results are plotted and compared with numerical results on this unstable displacement process available in the literature. The agreement is found to be excellent. In particular, results show that the non-Newtonian case (i.e., Oldroyd-B) is more unstable than the Newtonian case. Supported by an NPRP Grant # 08-777-1-141 from the Qatar National Research Fund (a member of the Qatar Foundation). The statements made herein are solely the responsibility of the author.

  12. Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants.

    PubMed

    Cho, Hyun-Jung; Lee, Seung Hee; Yoo, James J; Shon, Yun-Hee

    2014-04-01

    A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me2SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me2SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me2SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3weeks) and long-term (1year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media. PMID:24530510

  13. A phase 1 study of the bispecific anti-CD30/CD16A antibody construct AFM13 in patients with relapsed or refractory Hodgkin lymphoma

    PubMed Central

    Rothe, Achim; Sasse, Stephanie; Topp, Max S.; Eichenauer, Dennis A.; Hummel, Horst; Reiners, Katrin S.; Dietlein, Markus; Kuhnert, Georg; Kessler, Joerg; Buerkle, Carolin; Ravic, Miroslav; Knackmuss, Stefan; Marschner, Jens-Peter; Pogge von Strandmann, Elke; Borchmann, Peter

    2015-01-01

    AFM13 is a bispecific, tetravalent chimeric antibody construct (TandAb) designed for the treatment of CD30-expressing malignancies. AFM13 recruits natural killer (NK) cells via binding to CD16A as immune effector cells. In this phase 1 dose-escalation study, 28 patients with heavily pretreated relapsed or refractory Hodgkin lymphoma received AFM13 at doses of 0.01 to 7 mg/kg body weight. Primary objectives were safety and tolerability. Secondary objectives included pharmacokinetics, antitumor activity, and pharmacodynamics. Adverse events were generally mild to moderate. The maximum tolerated dose was not reached. Pharmacokinetics assessment revealed a half-life of up to 19 hours. Three of 26 evaluable patients achieved partial remission (11.5%) and 13 patients achieved stable disease (50%), with an overall disease control rate of 61.5%. AFM13 was also active in brentuximab vedotin–refractory patients. In 13 patients who received doses of ≥1.5 mg/kg AFM13, the overall response rate was 23% and the disease control rate was 77%. AFM13 treatment resulted in a significant NK-cell activation and a decrease of soluble CD30 in peripheral blood. In conclusion, AFM13 represents a well-tolerated, safe, and active targeted immunotherapy of Hodgkin lymphoma. A phase 2 study is currently planned to optimize the dosing schedule in order to further improve the therapeutic efficacy. This phase 1 study was registered at www.clinicaltrials.gov as #NCT01221571. PMID:25887777

  14. Time-dependent surface adhesive force and morphology of RBC measured by AFM.

    PubMed

    Wu, Yangzhe; Hu, Yi; Cai, Jiye; Ma, Shuyuan; Wang, Xiaoping; Chen, Yong; Pan, Yunlong

    2009-04-01

    Atomic force microscopy (AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains, potentially providing legal medical experts useful information for forensic investigation. In this study, the time-dependent, morphological changes of red blood cells (RBC) under three different conditions (including controlled, room-temperature condition, uncontrolled, outdoor-environmental condition, and controlled, low-temperature condition) were observed by AFM, as well as the cellular viscoelasticity via force-vs-distance curve measurements. Firstly, the data indicate that substrate types have different effects on cellular morphology of RBC. RBC presented the typical biconcave shape on mica, whereas either the biconcave shape or flattened shape was evident on glass. The mean volume of RBCs on mica was significantly larger than that of cells on glass. Surprisingly, the adhesive property of RBC membrane surfaces was substrate type-independent (the adhesive forces were statistically similar on glass and mica). With time lapse, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition. Under the controlled low-temperature condition, however, the changes in cell volume occurred mainly due to the collapse of RBCs, and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC. Taken together, the AFM detections on the time-dependent, substrate type-dependent, environment (temperature/humidity)-dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in forensic medicine or investigations, e.g., estimating age of bloodstain or death time. PMID:19019689

  15. Atomic force microscopy combined with optical tweezers (AFM/OT)

    NASA Astrophysics Data System (ADS)

    Pierini, F.; Zembrzycki, K.; Nakielski, P.; Pawłowska, S.; Kowalewski, T. A.

    2016-02-01

    The role of mechanical properties is essential to understand molecular, biological materials, and nanostructures dynamics and interaction processes. Atomic force microscopy (AFM) is the most commonly used method of direct force evaluation, but due to its technical limitations this single probe technique is unable to detect forces with femtonewton resolution. In this paper we present the development of a combined atomic force microscopy and optical tweezers (AFM/OT) instrument. The focused laser beam, on which optical tweezers are based, provides us with the ability to manipulate small dielectric objects and to use it as a high spatial and temporal resolution displacement and force sensor in the same AFM scanning zone. We demonstrate the possibility to develop a combined instrument with high potential in nanomechanics, molecules manipulation and biological studies. AFM/OT equipment is described and characterized by studying the ability to trap dielectric objects and quantifying the detectable and applicable forces. Finally, optical tweezers calibration methods and instrument applications are given.

  16. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ.

    PubMed

    Thi-Kim Vu, Hanh; Rink, Jochen C; McKinney, Sean A; McClain, Melainia; Lakshmanaperumal, Naharajan; Alexander, Richard; Sánchez Alvarado, Alejandro

    2015-01-01

    Cystic kidney diseases (CKDs) affect millions of people worldwide. The defining pathological features are fluid-filled cysts developing from nephric tubules due to defective flow sensing, cell proliferation and differentiation. The underlying molecular mechanisms, however, remain poorly understood, and the derived excretory systems of established invertebrate models (Caenorhabditis elegans and Drosophila melanogaster) are unsuitable to model CKDs. Systematic structure/function comparisons revealed that the combination of ultrafiltration and flow-associated filtrate modification that is central to CKD etiology is remarkably conserved between the planarian excretory system and the vertebrate nephron. Consistently, both RNA-mediated genetic interference (RNAi) of planarian orthologues of human CKD genes and inhibition of tubule flow led to tubular cystogenesis that share many features with vertebrate CKDs, suggesting deep mechanistic conservation. Our results demonstrate a common evolutionary origin of animal excretory systems and establish planarians as a novel and experimentally accessible invertebrate model for the study of human kidney pathologies. PMID:26057828

  17. Direct Visualization of the Hydration Layer on Alumina Nanoparticles with the Fluid Cell STEM in situ

    PubMed Central

    Firlar, Emre; Çınar, Simge; Kashyap, Sanjay; Akinc, Mufit; Prozorov, Tanya

    2015-01-01

    Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions. We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. Our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles. PMID:25996055

  18. Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3-dioxygenase1

    PubMed Central

    Romani, Rita; Pirisinu, Irene; Calvitti, Mario; Pallotta, Maria Teresa; Gargaro, Marco; Bistoni, Giovanni; Vacca, Carmine; Di Michele, Alessandro; Orabona, Ciriana; Rosati, Jessica; Pirro, Matteo; Giovagnoli, Stefano; Matino, Davide; Prontera, Paolo; Rosi, Gabriella; Grohmann, Ursula; Talesa, Vincenzo N; Donti, Emilio; Puccetti, Paolo; Fallarino, Francesca

    2015-01-01

    Although human amniotic fluid does contain different populations of foetal-derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second-trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)-γ, including induction of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN-γ–treated fHASCs caused significantly decreased T-cell proliferation and increased frequency in CD4+ CD25+ FOXP3+ regulatory T cells. Both effects required an intact IDO1 function and were cell contact-independent. An unprecedented finding in our study was that purified vesicles from IFN-γ–treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC-like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4+ CD25+ Foxp3+ T cells in graft-draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo. PMID:25783564

  19. End plate assembly having a two-phase fluid-filled bladder and method for compressing a fuel cell stack

    DOEpatents

    Carlstrom, Jr., Charles M.

    2001-01-01

    An end plate assembly is disclosed for use in a fuel cell assembly in which the end plate assembly includes a housing having a cavity, and a bladder receivable in the cavity and engageable with the fuel cell stack. The bladder includes a two-phase fluid having a liquid portion and a vapor portion. Desirably, the two-phase fluid has a vapor pressure between about 100 psi and about 600 psi at a temperature between about 70 degrees C. to about 110 degrees C.

  20. Cerebrospinal fluid T-regulatory cells recognize Borrelia burgdorferi NAPA in chronic Lyme borreliosis.

    PubMed

    Amedei, A; Codolo, G; Ozolins, D; Ballerini, C; Biagioli, T; Jaunalksne, I; Zilevica, A; D Elios, S; De Bernard, M; D' Elios, M M

    2013-01-01

    The NapA protein of B. burgdorferi is essential for the persistence of spirochetes in ticks. One of the most intriguing aspects of NapA is its potential to interfere with the host immune system. Here, we investigated the role of the acquired immune responses induced by NapA in the cerebrospinal fluids (CSF) of patients with chronic Lyme borreliosis. We evaluated the cytokine profile induced in microglia cells and CSF T cells following NapA stimulation. We report here that NapA induced a regulatory T (Treg) response in the CSF of patients with chronic Lyme borreliosis and it is able to expand this suppressive response by promoting the production of TGF-beta and IL-10 by microglia cells. Collectively, these data strongly support a central role of NapA in promoting both Treg response and immune suppression in the CSF of patients with chronic Lyme borreliosis and suggest that NapA and the Treg pathway may represent novel therapeutic targets for the prevention and treatment of the disease. PMID:24355226

  1. New miRNAs network in human mesenchymal stem cells derived from skin and amniotic fluid.

    PubMed

    Lazzarini, R; Sorgentoni, G; Caffarini, M; Sayeed, M A; Olivieri, F; Di Primio, R; Orciani, M

    2016-09-01

    Mesenchymal stem cells (MSCs), isolated from different adult sources, have great appeal for therapeutic applications due to their simple isolation, extensive expansion potential, and high differentiative potential.In our previous studies we isolated MSCs form amniotic fluid (AF-MSCs) and skin (S-MSCs) and characterized them according to their phenotype, pluripotency, and mRNA/microRNAs (miRNAs) profiling using Card A from Life Technologies.Here, we enlarge the profiling of AF-MCSs and S-MSCs to the more recently discovered miRNAs (Card B by Life Technologies) to identify the miRNAs putative target genes and the relative signaling pathways. Card B, in fact, contains miRNAs whose role and target are not yet elucidated.The expression of the analyzed miRNAs is changing between S-MSCs and AF-MSCs, indicating that these two types of MSCs show differences potentially related to their source. Interestingly, the pathways targeted by the miRNAS deriving from Card B are the same found during the analysis of miRNAs from Card A.This result confirms the key role played by WNT and TGF-β pathways in stem cell fate, underlining as other miRNAs partially ignored up to now deserve to be reconsidered. In addition, this analysis allows including Adherens junction pathways among the mechanisms finely regulated in stem cell behavior. PMID:26684628

  2. Amniotic Fluid Stem Cells from EGFP Transgenic Mice Attenuate Hyperoxia-Induced Acute Lung Injury

    PubMed Central

    Lai, Cheng-Wei; Yen, Chih-Ching; Lee, Kun-Hsiung; Wu, Shinn-Chih; Chen, Chuan-Mu

    2013-01-01

    High concentrations of oxygen aggravate the severity of lung injury in patients requiring mechanical ventilation. Although mesenchymal stem cells have been shown to effectively attenuate various injured tissues, there is limited information regarding a role for amniotic fluid stem cells (AFSCs) in treating acute lung injury. We hypothesized that intravenous delivery of AFSCs would attenuate lung injury in an experimental model of hyperoxia-induced lung injury. AFSCs were isolated from EGFP transgenic mice. The in vitro differentiation, surface markers, and migration of the AFSCs were assessed by specific staining, flow cytometry, and a co-culture system, respectively. The in vivo therapeutic potential of AFSCs was evaluated in a model of acute hyperoxia-induced lung injury in mice. The administration of AFSCs significantly reduced the hyperoxia-induced pulmonary inflammation, as reflected by significant reductions in lung wet/dry ratio, neutrophil counts, and the level of apoptosis, as well as reducing the levels of inflammatory cytokine (IL-1β, IL-6, and TNF-α) and early-stage fibrosis in lung tissues. Moreover, EGFP-expressing AFSCs were detected and engrafted into a peripheral lung epithelial cell lineage by fluorescence microscopy and DAPI stain. Intravenous administration of AFSCs may offer a new therapeutic strategy for acute lung injury (ALI), for which efficient treatments are currently unavailable. PMID:24040409

  3. Peritoneal dialysis fluid activates calcium signaling and apoptosis in mesothelial cells.

    PubMed

    Boccellino, Mariarosaria; La Porta, Raffaele; Coppola, Mario; Petronella, Pasquale; Freda, Fulvio; Calderaro, Vincenzo; Quagliuolo, Lucio

    2013-01-01

    A larger diffusion of peritoneal dialysis (PD) is limited by the progressive deterioration of the dialysis membrane structure and function, characterized in vitro and in vivo by mesothelial cell loss and closely related to the use of bioincompatible dialysis solutions. The apoptosis rate of rat and human mesothelial cells incubated in commercial PD fluid (PDF, 4.25 g/dL dextrose) became significant as early as 1 h after PDF addition and reached a plateau at 4-5 h. This pattern was unchanged after exposure to 1.5 g/dL dextrose PDF or freshly prepared PDF, indicating that effects were independent on the dextrose strength and manufacturing procedures but strictly dependent on PDF composition. Molecular studies revealed that PDF exposure inactivated the physiological volume recovery from hypertonic shrinkage, accompanied by an abnormal Ca(2+) signaling: a progressive intracellular Ca(2+) ([Ca(2+)](i)) rise resulting from an increased Ca(2+) entry. PDF also affected cytoskeleton integrity: early dissolution of actin filaments occurred well before the appearance of typical apoptosis features. Lastly, the PDF dependent apoptosis was almost completely prevented by the contemporary Ca(2+) concentration decrease and K(+) addition. This study suggests that the PDF dependent apoptosis arises from the extreme volume perturbations in mesothelial cells, turned out unable to regulate their volume back once exposed to a hyperosmolal medium containing high Ca(2+) levels in the absence of K(+), such PDF. PMID:23100160

  4. Nanoscale structural features determined by AFM for single virus particles.

    PubMed

    Chen, Shu-wen W; Odorico, Michael; Meillan, Matthieu; Vellutini, Luc; Teulon, Jean-Marie; Parot, Pierre; Bennetau, Bernard; Pellequer, Jean-Luc

    2013-11-21

    In this work, we propose "single-image analysis", as opposed to multi-image averaging, for extracting valuable information from AFM images of single bio-particles. This approach allows us to study molecular systems imaged by AFM under general circumstances without restrictions on their structural forms. As feature exhibition is a resolution correlation, we have performed AFM imaging on surfaces of tobacco mosaic virus (TMV) to demonstrate variations of structural patterns with probing resolution. Two AFM images were acquired with the same tip at different probing resolutions in terms of pixel width, i.e., 1.95 and 0.49 nm per pixel. For assessment, we have constructed an in silico topograph based on the three-dimensional crystal structure of TMV as a reference. The prominent artifacts observed in the AFM-determined shape of TMV were attributed to tip convolutions. The width of TMV rod was systematically overestimated by ~10 nm at both probing resolutions of AFM. Nevertheless, the effects of tip convolution were less severe in vertical orientation so that the estimated height of TMV by AFM imaging was in close agreement with the in silico X-ray topograph. Using dedicated image processing algorithms, we found that at low resolution (i.e., 1.95 nm per pixel), the extracted surface features of TMV can be interpreted as a partial or full helical repeat (three complete turns with ~7.0 nm in length), while individual protein subunits (~2.5 nm) were perceivable only at high resolution. The present study shows that the scales of revealed structural features in AFM images are subject to both probing resolution and processing algorithms for image analysis. PMID:24056758

  5. The Conductance of Nanotubes Deformed by the AFM Tip

    NASA Technical Reports Server (NTRS)

    Svizhenko, Alexei; Maiti, Amitesh; Anantram, M. P.

    2003-01-01

    The conductance drop under AFM-tip deformation can be explained by stretching of the tube length. NT sensors can be built utilizing uniform stretching. Single sp3 bond cross section cannot block electrons, because another conducting path may exist. AFM tip which forms sp3 bonds with the tube will decrease conductance. In the "table experiment" a conductance drop of 2 orders of magnitude happened only after some bonds were broken.

  6. Characterization of the interaction between AFM tips and surface nanobubbles.

    PubMed

    Walczyk, Wiktoria; Schönherr, Holger

    2014-06-24

    While the presence of gaseous enclosures observed at various solid-water interfaces, the so-called "surface nanobubles", has been confirmed by many groups in recent years, their formation, properties, and stability have not been convincingly and exhaustively explained. Here we report on an atomic force microscopy (AFM) study of argon nanobubbles on highly oriented pyrolitic graphite (HOPG) in water to elucidate the properties of nanobubble surfaces and the mechanism of AFM tip-nanobubble interaction. In particular, the deformation of the nanobubble-water interface by the AFM tip and the question whether the AFM tip penetrates the nanobubble during scanning were addressed by this combined intermittent contact (tapping) mode and force volume AFM study. We found that the stiffness of nanobubbles was smaller than the cantilever spring constant and comparable with the surface tension of water. The interaction with the AFM tip resulted in severe quasi-linear deformation of the bubbles; however, in the case of tip-bubble attraction, the interface deformed toward the tip. We tested two models of tip-bubble interaction, namely, the capillary force and the dynamic interaction model, and found, depending on the tip properties, good agreement with experimental data. The results showed that the tip-bubble interaction strength and the magnitude of the bubble deformation depend strongly on tip and bubble geometry and on tip and substrate material, and are very sensitive to the presence of contaminations that alter the interfacial tension. In particular, nanobubbles interacted differently with hydrophilic and hydrophobic AFM tips, which resulted in qualitatively and quantitatively different force curves measured on the bubbles in the experiments. To minimize bubble deformation and obtain reliable AFM results, nanobubbles must be measured with a sharp hydrophilic tip and with a cantilever having a very low spring constant in a contamination-free system. PMID:24856074

  7. A two-dimensional simulation of the GEC RF reference cell using a hybrid fluid-Monte Carlo method

    SciTech Connect

    Pak, H.; Riley, M.E.

    1992-12-01

    A two-dimensional fluid-Monte Carlo hybrid model is used to simulate the GEC reference cell. The 2-D model assumes azimuthal symmetry and accounts for the ground shield about the electrodes as well as the grounded chamber walls. The hybrid model consists of a Monte Carlo method for generating rates and a fluid model for transporting electrons and ions. In the fluid model, the electrons are transported using the continuity equation; and the electric fields are solved self-consistently using Poisson`s equation. The Monte Carlo model transports electrons using the fluid-generated periodic electric field. The ionization rates are then obtained using the electron energy distribution function. An averaging method is used to speed the solution by transporting the ions in a time-averaged electric field with a corrected ambipolar-type diffusion. The simulation switches between the conventional and the averaging fluid model. Typically, the simulation runs from 10`s to 100`s of averaging fluid cycles before reentering the conventional fluid model for 10`s of cycles. Speed increases of a factor of 100 are possible.

  8. Labile iron in cells and body fluids: physiology, pathology, and pharmacology

    PubMed Central

    Cabantchik, Zvi Ioav

    2014-01-01

    In living systems iron appears predominantly associated with proteins, but can also be detected in forms referred as labile iron, which denotes the combined redox properties of iron and its amenability to exchange between ligands, including chelators. The labile cell iron (LCI) composition varies with metal concentration and substances with chelating groups but also with pH and the medium redox potential. Although physiologically in the lower μM range, LCI plays a key role in cell iron economy as cross-roads of metabolic pathways. LCI levels are continually regulated by an iron-responsive machinery that balances iron uptake versus deposition into ferritin. However, LCI rises aberrantly in some cell types due to faulty cell utilization pathways or infiltration by pathological iron forms that are found in hemosiderotic plasma. As LCI attains pathological levels, it can catalyze reactive O species (ROS) formation that, at particular threshold, can surpass cellular anti-oxidant capacities and seriously damage its constituents. While in normal plasma and interstitial fluids, virtually all iron is securely carried by circulating transferrin (Tf; that renders iron essentially non-labile), in systemic iron overload (IO), the total plasma iron binding capacity is often surpassed by a massive iron influx from hyperabsorptive gut or from erythrocyte overburdened spleen and/or liver. As plasma Tf approaches iron saturation, labile plasma iron (LPI) emerges in forms that can infiltrate cells by unregulated routes and raise LCI to toxic levels. Despite the limited knowledge available on LPI speciation in different types and degrees of IO, LPI measurements can be and are in fact used for identifying systemic IO and for initiating/adjusting chelation regimens to attain full-day LPI protection. A recent application of labile iron assay is the detection of labile components in intravenous iron formulations per se as well as in plasma (LPI) following parenteral iron administration

  9. Topology optimization of adaptive fluid-actuated cellular structures with arbitrary polygonal motor cells

    NASA Astrophysics Data System (ADS)

    Lv, Jun; Tang, Liang; Li, Wenbo; Liu, Lei; Zhang, Hongwu

    2016-05-01

    This paper mainly focuses on the fast and efficient design method for plant bioinspired fluidic cellular materials and structures composed of polygonal motor cells. Here we developed a novel structural optimization method with arbitrary polygonal coarse-grid elements based on multiscale finite element frameworks. The fluidic cellular structures are meshed with irregular polygonal coarse-grid elements according to their natural size and the shape of the imbedded motor cells. The multiscale base functions of solid displacement and hydraulic pressure are then constructed to bring the small-scale information of the irregular motor cells to the large-scale simulations on the polygonal coarse-grid elements. On this basis, a new topology optimization method based on the resulting polygonal coarse-grid elements is proposed to determine the optimal distributions or number of motor cells in the smart cellular structures. Three types of optimization problems are solved according to the usages of the fluidic cellular structures. Firstly, the proposed optimization method is utilized to minimize the system compliance of the load-bearing fluidic cellular structures. Second, the method is further extended to design biomimetic compliant actuators of the fluidic cellular materials due to the fact that non-uniform volume expansions of fluid in the cells can induce elastic action. Third, the optimization problem focuses on the weight minimization of the cellular structure under the constraints for the compliance of the whole system. Several representative examples are investigated to validate the effectiveness of the proposed polygon-based topology optimization method of the smart materials.

  10. No detection of BK virus, JC virus, KI, WU and Merkel cell polyomaviruses in cerebrospinal fluid of patients with neurological complications after hematopoetic stem cell transplantation.

    PubMed

    Rubin, J; Giraud, G; Priftakis, P; Wide, K; Gustafsson, B; Ramqvist, T; Dalianis, T

    2011-10-01

    Neurological complications, often due to viral reactivation, after allogeneic hematopoetic stem cell transplantation (HSCT) are associated with increased mortality. Here, cerebrospinal fluid from 20 HSCT patients with neurological symptoms were analyzed and found to be negative by PCR for BK virus, JC virus, KI, WU and Merkel cell polyomavirus DNA. PMID:21965766

  11. Development & experimental validation of a SINDA/FLUINT thermal/fluid/electrical model of a multi-tube AMTEC cell

    NASA Astrophysics Data System (ADS)

    Hendricks, Terry J.; Borkowski, Chris A.; Huang, Chendong

    1998-01-01

    AMTEC (Alkali Metal Thermal-to-Electric Conversion) cell development has received increased attention and funding in the space power community because of several desirable performance characteristics compared to current radioisotope thermoelectric generation and solar photovoltaic (PV) power generation. AMTEC cell development is critically dependent upon the ability to predict thermal, fluid dynamic and electrical performance of an AMTEC cell which has many complex thermal, fluid dynamic and electrical processes and interactions occurring simultaneously. Development of predictive capability is critical to understanding the complex processes and interactions within the AMTEC cell, and thereby creating the ability to design high-performance, cost-effective AMTEC cells. A flexible, sophisticated thermal/fluid/electrical model of an operating AMTEC cell has been developed using the SINDA/FLUINT analysis software. This model can accurately simulate AMTEC cell performance at any hot side and cold side temperature combination desired, for any voltage and current conditions, and for a broad range of cell design parameters involving the cell dimensions, current collector and electrode design, electrode performance parameters, and cell wall and thermal shield emissivity. The model simulates the thermal radiation network within the AMTEC cell using RadCAD thermal radiation analysis; hot side, cold side and cell wall conductive and radiative coupling; BASE (Beta Alumina Solid Electrode) tube electrochemistry, including electrode over-potentials; the fluid dynamics of the low-pressure sodium vapor flow to the condenser and liquid sodium flow in the wick; sodium condensation at the condenser; and high-temperature sodium evaporation in the wick. The model predicts the temperature profiles within the AMTEC cell walls, the BASE tube temperature profiles, the sodium temperature profile in the artery return, temperature profiles in the evaporator, thermal energy flows throughout the

  12. BOREAS AFM-12 1-km AVHRR Seasonal Land Cover Classification

    NASA Technical Reports Server (NTRS)

    Steyaert, Lou; Hall, Forrest G.; Newcomer, Jeffrey A. (Editor); Knapp, David E. (Editor); Loveland, Thomas R.; Smith, David E. (Technical Monitor)

    2000-01-01

    features such as fens, bogs, and small water bodies. Field observations and comparisons with Landsat Thematic Mapper (TM) suggest a minimum effective resolution of these land cover classes in the range of three to four kilometers, in part, because of the daily to monthly compositing process. In general, potential accuracy limitations are mitigated by the use of conservative parameterization rules such as aggregation of predominant land cover classes within minimum horizontal grid cell sizes of ten kilometers. The AFM-12 one-kilometer AVHRR seasonal land cover classification data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  13. Modeling the Interaction between AFM Tips and Pinned Surface Nanobubbles.

    PubMed

    Guo, Zhenjiang; Liu, Yawei; Xiao, Qianxiang; Schönherr, Holger; Zhang, Xianren

    2016-01-26

    Although the morphology of surface nanobubbles has been studied widely with different AFM modes, AFM images may not reflect the real shapes of the nanobubbles due to AFM tip-nanobubble interactions. In addition, the interplay between surface nanobubble deformation and induced capillary force has not been well understood in this context. In our work we used constraint lattice density functional theory to investigate the interaction between AFM tips and pinned surface nanobubbles systematically, especially concentrating on the effects of tip hydrophilicity and shape. For a hydrophilic tip contacting a nanobubble, its hydrophilic nature facilitates its departure from the bubble surface, displaying a weak and intermediate-range attraction. However, when the tip squeezes the nanobubble during the approach process, the nanobubble shows an elastic effect that prevents the tip from penetrating the bubble, leading to a strong nanobubble deformation and repulsive interactions. On the contrary, a hydrophobic tip can easily pierce the vapor-liquid interface of the nanobubble during the approach process, leading to the disappearance of the repulsive force. In the retraction process, however, the adhesion between the tip and the nanobubble leads to a much stronger lengthening effect on nanobubble deformation and a strong long-range attractive force. The trends of force evolution from our simulations agree qualitatively well with recent experimental AFM observations. This favorable agreement demonstrates that our model catches the main intergradient of tip-nanobubble interactions for pinned surface nanobubbles and may therefore provide important insight into how to design minimally invasive AFM experiments. PMID:26751634

  14. Formulation, Implementation and Validation of a Two-Fluid model in a Fuel Cell CFD Code

    SciTech Connect

    Kunal Jain, Vernon Cole, Sanjiv Kumar and N. Vaidya

    2008-11-01

    Water management is one of the main challenges in PEM Fuel Cells. While water is essential for membrane electrical conductivity, excess liquid water leads to ooding of catalyst layers. Despite the fact that accurate prediction of two-phase transport is key for optimal water management, understanding of the two-phase transport in fuel cells is relatively poor. Wang et. al. [1], [2] have studied the two-phase transport in the channel and diffusion layer separately using a multiphase mixture model. The model fails to accurately predict saturation values for high humidity inlet streams. Nguyen et. al. [3] developed a two-dimensional, two-phase, isothermal, isobaric, steady state model of the catalyst and gas diffusion layers. The model neglects any liquid in the channel. Djilali et. al. [4] developed a three-dimensional two-phase multicomponent model. The model is an improvement over previous models, but neglects drag between the liquid and the gas phases in the channel. In this work, we present a comprehensive two- fluid model relevant to fuel cells. Models for two-phase transport through Channel, Gas Diffusion Layer (GDL) and Channel-GDL interface, are discussed. In the channel, the gas and liquid pressures are assumed to be same. The surface tension effects in the channel are incorporated using the continuum surface force (CSF) model. The force at the surface is expressed as a volumetric body force and added as a source to the momentum equation. In the GDL, the gas and liquid are assumed to be at different pressures. The difference in the pressures (capillary pressure) is calculated using an empirical correlations. At the Channel-GDL interface, the wall adhesion affects need to be taken into account. SIMPLE-type methods recast the continuity equation into a pressure-correction equation, the solution of which then provides corrections for velocities and pressures. However, in the two-fluid model, the presence of two phasic continuity equations gives more freedom and

  15. Cerebrospinal fluid white cell count: discriminatory or otherwise for enteroviral meningitis in infants and young children?

    PubMed

    Tan, Natalie Woon Hui; Lee, Elis Yuexian; Khoo, Gloria Mei Chin; Tee, Nancy Wen Sim; Krishnamoorthy, Subramania; Choong, Chew Thye

    2016-04-01

    Non-polio enteroviruses (EV) are the most common viruses causing aseptic meningitis in children. We aim to evaluate the cerebrospinal fluid (CSF) characteristics of neonates and children with EV meningitis with a view to determine whether it could be discriminatory or otherwise in making a positive diagnosis. We performed a 3-year (July 2008-July 2011) retrospective study of children ≤16 years, treated at a tertiary children's hospital, with positive CSF EV polymerase chain reaction (PCR) and negative blood and CSF bacterial cultures. A total of 206 children were studied. The median CSF white cell count was 79 cells/mm(3) (range 0-4608 cells/mm(3)). CSF pleocytosis was observed in 99/150 (66%) aged ≤90 days, 3/4 (75%) aged 90 days-1 year, and 49/52 (94%) children ≥3 years. There was a huge variability in CSF pleocytosis in infants ≤90 days, where 34% of them had no pleocytosis, while in 66%, a wide range of pleocytosis that might even suggest bacterial meningitis was noted. CSF red cells were low, and protein or sugar values were not discriminatory. CSF pleocytosis in relation to increasing age was found to be statistically significant (p < 0.001). Early lumbar puncture within 48 h of symptoms and absence of CSF pleocytosis was also statistically significant (p = 0.039). CSF pleocytosis in EV meningitis is commoner in older children. As there was a huge variability in CSF pleocytosis in infants ≤90 days particularly, CSF analysis including EV PCR could avoid unnecessary antibiotic therapy. PMID:26463525

  16. Two-dimensional fluid-filled closed-cell cellular solid as an acoustic metamaterial with negative index

    NASA Astrophysics Data System (ADS)

    Dorodnitsyn, V.; Van Damme, B.

    2016-04-01

    A concept for acoustic metamaterials consisting of a cellular medium with fluid-filled cells is fabricated and studied experimentally. In such a system, the fluid and solid structure explicitly interact, and elastic wave propagation is coupled to both phases. Focusing here on shear wave behavior, we confirm previous numerical studies in three steps. We first measure the material deformations pertaining to three qualitatively different shear wave modes in the frequency range below 3.5 kHz. We then measure the group velocity and demonstrate that, within a certain frequency interval, the group and phase velocity have opposite signs. This shows that the system acts as a negative-index metamaterial. Finally, we confirm the presence of band gaps due to the locally resonant behavior of the cell walls. The demonstrated concept of a closed, fluid-filled cellular material as an acoustic metamaterial opens a wide space for applications.

  17. A Shearing-Stretching Device That Can Apply Physiological Fluid Shear Stress and Cyclic Stretch Concurrently to Endothelial Cells.

    PubMed

    Meza, Daphne; Abejar, Louie; Rubenstein, David A; Yin, Wei

    2016-03-01

    Endothelial cell (EC) morphology and functions can be highly impacted by the mechanical stresses that the cells experience in vivo. In most areas in the vasculature, ECs are continuously exposed to unsteady blood flow-induced shear stress and vasodilation-contraction-induced tensile stress/strain simultaneously. Investigations on how ECs respond to combined shear stress and tensile strain will help us to better understand how an altered mechanical environment affects EC mechanotransduction, dysfunction, and associated cardiovascular disease development. In the present study, a programmable shearing and stretching device that can apply dynamic fluid shear stress and cyclic tensile strain simultaneously to cultured ECs was developed. Flow and stress/strain conditions in the device were simulated using a fluid structure interaction (FSI) model. To characterize the performance of this device and the effect of combined shear stress-tensile strain on EC morphology, human coronary artery ECs (HCAECs) were exposed to concurrent shear stress and cyclic tensile strain in the device. Changes in EC morphology were evaluated through cell elongation, cell alignment, and cell junctional actin accumulation. Results obtained from the numerical simulation indicated that in the "in-plane" area of the device, both fluid shear stress and biaxial tensile strain were uniform. Results obtained from the in vitro experiments demonstrated that shear stress, alone or combined with cyclic tensile strain, induced significant cell elongation. While biaxial tensile strain alone did not induce any appreciable change in EC elongation. Fluid shear stress and cyclic tensile strain had different effects on EC actin filament alignment and accumulation. By combining various fluid shear stress and cyclic tensile strain conditions, this device can provide a physiologically relevant mechanical environment to study EC responses to physiological and pathological mechanical stimulation. PMID:26810848

  18. Assembly of live micro-organisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments

    NASA Astrophysics Data System (ADS)

    Dague, E.; Jauvert, E.; Laplatine, L.; Viallet, B.; Thibault, C.; Ressier, L.

    2011-09-01

    Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.

  19. Volume-of-fluid simulations of bubble dynamics in a vertical Hele-Shaw cell

    NASA Astrophysics Data System (ADS)

    Wang, Xue; Klaasen, Bart; Degrève, Jan; Mahulkar, Amit; Heynderickx, Geraldine; Reyniers, Marie-Françoise; Blanpain, Bart; Verhaeghe, Frederik

    2016-05-01

    Bubbles in confined geometries serve an important role for industrial operations involving bubble-liquid interactions. However, high Reynolds number bubble dynamics in confined flows are still not well understood due to experimental challenges. In the present paper, combined experimental and numerical methods are used to provide a comprehensive insight into these dynamics. The bubble behaviour in a vertical Hele-Shaw cell is investigated experimentally with a fully wetting liquid for a variety of gap thicknesses. A numerical model is developed using the volume of fluid method coupled with a continuum surface force model and a wall friction model. The developed model successfully simulates the dynamics of a bubble under the present experimental conditions and shows good agreement between experimental and simulation results. It is found that with an increased spacing between the cell walls, the bubble shape changes from oblate ellipsoid and spherical-cap to more complicated shapes, while the bubble path changes from only rectilinear to a combination of oscillating and rectilinear; the bubble drag coefficient decreases and this results in a higher bubble velocity caused by a lower pressure exerted on the bubble; the wake boundary and wake length evolve gradually accompanied by vortex formation and shedding.

  20. Granulosa Cell Apoptosis Induced by a Novel FSH Binding Inhibitory Peptide From Human Ovarian Follicular Fluid

    PubMed Central

    Chitnis, Swati S.; Navlakhe, Rajshri M.; Shinde, Gayatri C.; Barve, Sharmila J.; D'Souza, Serena; Mahale, Smita D.; Nandedkar, Tarala D.

    2008-01-01

    Pituitary gonadotropins, follicle-stimulating hormone and luteinizing hormone, are the key regulators of ovarian folliculogenesis; these are known to be directly or indirectly modulated by many intraovarian factors. Our group has identified and studied one such novel peptide from human ovarian follicular fluid. Its partial N-terminal eight amino acid sequence has been deduced, referred to as octapeptide (OP). OP induces follicular atresia in mice and interferes with normal ovarian function in non-human primates, this action being similar to the native peptide. Thus, in this study, an attempt has been made to elucidate the mechanism of action of the synthetic OP by studying the pathway of follicular atresia in mouse ovary. Changes in granulosa cells were studied using various apoptotic markers by flow cytometry and immunohistochemistry. An increase in apoptotic cell population in atretic- and peptide-treated groups was observed compared with normal controls. Interestingly, both these groups exhibited differences in the apoptotic pathway. Results showed that the mitochondrial pathway was predominant in the atretic group, whereas the Fas-FasL pathway was predominant in the peptide-treated groups. The ultrastructural study also showed apoptotic changes in the OP-treated and atretic groups; the pattern of apoptosis differed at the subcellular level. (J Histochem Cytochem 56:961–968, 2008) PMID:18645207

  1. An adaptive cut-cell method for animal-locomotion fluid mechanics

    NASA Astrophysics Data System (ADS)

    Pederzani, Jean-Noel; Haj-Hariri, H.

    2011-11-01

    In this work we present a numerical method for solving the incompressible Navier-Stokes equation for biomimetic fluid-structure interaction problems. The method is designed to study the flow generated by interaction with arbitrarily complex motion of a self-propelling animal. We consider the specific case of a manta ray. The method combines the embedded-boundary (or cut-cell) method for complex geometry with moving boundaries, and block-structured adaptive mesh refinement (AMR). The control volumes are formed by the intersection of the irregular boundary with Cartesian grid cells. These control volumes fit naturally within parallelizable, disjoint-block data structures, and permit dynamic AMR coarsening and refinement as the simulation progresses. We present two- and three-dimensional results to illustrate the accuracy of the method. Results are compared with experimental results for a flapping elliptical fin that mimics the natural motion of a manta ray. In particular the hydrodynamic signature of the vortex structure behind the fin is studied for its effect on swimming performance.

  2. AFM imaging reveals the tetrameric structure of the TRPM8 channel

    SciTech Connect

    Stewart, Andrew P.; Egressy, Kinga; Lim, Annabel; Edwardson, J. Michael

    2010-04-02

    Several members of the transient receptor potential (TRP) channel superfamily have been shown to assemble as tetramers. Here we have determined the subunit stoichiometry of the transient receptor potential M8 (TRPM8) channel using atomic force microscopy (AFM). TRPM8 channels were isolated from transfected cells, and complexes were formed between the channels and antibodies against a V5 epitope tag present on each subunit. The complexes were then subjected to AFM imaging. A frequency distribution of the molecular volumes of antibody decorated channels had a peak at 1305 nm{sup 3}, close to the expected size of a TRPM8 tetramer. The frequency distribution of angles between pairs of bound antibodies had two peaks, at 93{sup o} and 172{sup o}, confirming that the channel assembles as a tetramer. We suggest that this assembly pattern is common to all members of the TRP channel superfamily.

  3. Fluid imbalance

    MedlinePlus

    ... up in the body. This is called fluid overload (volume overload). This can lead to edema (excess fluid in ... Water imbalance; Fluid imbalance - dehydration; Fluid buildup; Fluid overload; Volume overload; Loss of fluids; Edema - fluid imbalance; ...

  4. Thermomechanical analysis of freezing-induced cell-fluid-matrix interactions in engineered tissues

    PubMed Central

    Han, Bumsoo; Teo, Ka Yaw; Ghosh, Soham; Dutton, J. Craig; Grinnell, Frederick

    2012-01-01

    Successful cryopreservation of functional engineered tissues (ETs) is significant to tissue engineering and regenerative medicine, but it is extremely challenging to develop a successful protocol because the effects of cryopreservation parameters on the post-thaw functionality of ETs are not well understood. Particularly, the effects on the microstructure of their extracellular matrix (ECM) have not been well studied, which determines many functional properties of the ETs. In this study, we investigated the effects of two key cryopreservation parameters – i) freezing temperature and corresponding cooling rate; and ii) the concentration of cryoprotective agent (CPA) on the ECM microstructure as well as the cellular viability. Using dermal equivalent as a model ET and DMSO as a model CPA, freezing-induced spatiotemporal deformation and post-thaw ECM microstructure of ETs was characterized while varying the freezing temperature and DMSO concentrations. The spatial distribution of cellular viability and the cellular actin cytoskeleton was also examined. The results showed that the tissue dilatation increased significantly with reduced freezing temperature (i.e., rapid freezing). A maximum limit of tissue deformation was observed for preservation of ECM microstructure, cell viability and cell-matrix adhesion. The dilatation decreased with the use of DMSO, and a freezing temperature dependent threshold concentration of DMSO was observed. The threshold DMSO concentration increased with lowering freezing temperature. In addition, an analysis was performed to delineate thermodynamic and mechanical components of freezing-induced tissue deformation. The results are discussed to establish a mechanistic understanding of freezing-induced cell-fluid-matrix interaction and phase change behavior within ETs in order to improve cryopreservation of ETs. PMID:23246556

  5. The human amniotic fluid stem cell secretome effectively counteracts doxorubicin-induced cardiotoxicity

    PubMed Central

    Lazzarini, Edoardo; Balbi, Carolina; Altieri, Paola; Pfeffer, Ulrich; Gambini, Elisa; Canepa, Marco; Varesio, Luigi; Bosco, Maria Carla; Coviello, Domenico; Pompilio, Giulio; Brunelli, Claudio; Cancedda, Ranieri; Ameri, Pietro; Bollini, Sveva

    2016-01-01

    The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB, and upregulation of the NF-kB controlled genes, Il6 and Cxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter, Abcb1b, and Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of Il6, Cxcl1 and Abcb1b, and prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity. PMID:27444332

  6. The human amniotic fluid stem cell secretome effectively counteracts doxorubicin-induced cardiotoxicity.

    PubMed

    Lazzarini, Edoardo; Balbi, Carolina; Altieri, Paola; Pfeffer, Ulrich; Gambini, Elisa; Canepa, Marco; Varesio, Luigi; Bosco, Maria Carla; Coviello, Domenico; Pompilio, Giulio; Brunelli, Claudio; Cancedda, Ranieri; Ameri, Pietro; Bollini, Sveva

    2016-01-01

    The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB, and upregulation of the NF-kB controlled genes, Il6 and Cxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter, Abcb1b, and Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation of Il6, Cxcl1 and Abcb1b, and prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity. PMID:27444332

  7. Nonlinear Evolution of Ion Acoustic Solitary Waves in Earth's Magnetosphere: Fluid and Particle-In-Cell Simulations

    NASA Astrophysics Data System (ADS)

    Kakad, A.; Kakad, B. A.; Omura, Y.

    2014-12-01

    In recent spacecraft observations, coherent electrostatic solitary wave (ESWs) structures are observed in various regions of the Earth's magnetosphere. Over the years, many researchers have attempted to model these observations in terms of electron/ion acoustic solitary waves by using nonlinear fluid theory/simulations. The ESW structures predicted by fluid models can be inadequate due to its inability in handling kinetic effects. To provide clear view on the application of the fluid and kinetic treatments in modeling the ESWs, we perform both fluid and particle-in-cell (PIC) simulations of ion acoustic solitary waves (IASWs) and estimate the quantitative differences in their characteristics like speed, amplitude, and width. It is noted that a long time evolution of Gaussian type perturbations in the equilibrium electron and ion densities generated the nonlinear IASW structures in both fluid and PIC simulations. The IASW structures represent vortices of trapped electrons in PIC simulations. We find that the number of trapped electrons in the wave potential is higher for the large amplitude IASW, which are generated by large-amplitude initial density perturbation (IDP). The present fluid and PIC simulation results are in close agreement for small amplitude IDPs, whereas for large IDPs they show discrepancy in the amplitude, width, and speed of the IASW, which is attributed to negligence of kinetic effects in the former approach. The speed of IASW in the fluid simulations increases with the increase of IASW amplitude, while the reverse tendency is seen in the PIC simulation. The present study suggests that the fluid treatment is appropriate to model the IASW observations when the magnitude of phase velocity of IASW is less than the ion acoustic (IA) speed obtained from their linear dispersion relation, whereas when it exceeds IA speed, it is necessary to include the kinetic effects in the model.

  8. AFM investigation of Martian soil simulants on micromachined Si substrates.

    PubMed

    Vijendran, S; Sykulska, H; Pike, W T

    2007-09-01

    The micro and nanostructures of Martian soil simulants with particles in the micrometre-size range have been studied using a combination of optical and atomic force microscopy (AFM) in preparation for the 2007 NASA Phoenix Mars Lander mission. The operation of an atomic force microscope on samples of micrometre-sized soil particles is a poorly investigated area where the unwanted interaction between the scanning tip and loose particles results in poor image quality and tip contamination by the sample. In order to mitigate these effects, etched silicon substrates with a variety of features have been used to facilitate the sorting and gripping of particles. From these experiments, a number of patterns were identified that were particularly good at isolating and immobilizing particles for AFM imaging. This data was used to guide the design of micromachined substrates for the Phoenix AFM. Both individual particles as well as aggregates were successfully imaged, and information on sizes, shapes and surface morphologies were obtained. This study highlights both the strengths and weaknesses of AFM for the potential in situ investigation of Martian soil and dust. Also presented are more general findings of the limiting operational constraints that exist when attempting the AFM of high aspect ratio particles with current technology. The performance of the final designs of the substrates incorporated on Phoenix will be described in a later paper. PMID:17760618

  9. Pt and Pt-Ru/Carbon Nanotube Nanocomposites Synthesized in Supercritical Fluid as Electrocatalysts for Low-Temperature Fuel Cells

    SciTech Connect

    Lin, Yuehe; Cui, Xiaoli; Wang, Jun; Yen, Clive; Wai, Chien M.

    2006-06-01

    In recent years, the use of supercritical fluids (SCFs) for the synthesis and processing of nanomaterials has proven to be a rapid, direct, and clean approach to develop nanomaterials and nanocomposites. The application of supercritical fluid technology can result in products (and processes) that are cleaner, less expensive, and of higher quality than those that are produced using conventional technologies and solvents. In this work, carbon nanotube (CNT)-supported Pt and Pt-Ru nanoparticles catalysts have been synthesized in supercritical carbon dioxide (scCO2). The experimental results demonstrate that Pt, Pt-Ru/CNT nanocomposites synthesized in supercritical carbon dioxide are effective electrocatalysts for low-temperature fuel cells.

  10. Flagellar Kinematics and Swimming Behavior of Algal Cells in Viscoelastic Fluids

    NASA Astrophysics Data System (ADS)

    Arratia, Paulo; Yang, Jing; Gollub, Jerry

    2013-11-01

    The motility behavior of microorganisms can be significantly affected by the rheology of their fluidic environment. In this talk, we experimentally investigate the effects of fluid elasticity on both the flagella kinematics and swimming dynamics of the microscopic alga Chlamydomonas reinhardtii. We find that the flagellar beating frequency and wave speed are both enhanced by fluid elasticity. Interestingly, the swimming speeds during the alga power and recovery strokes are enhanced by fluid elasticity for De>1. Despite such enhancements, however, the alga net forward speed is hindered by fluid elasticity by as much as 30% compared to Newtonian fluids of similar shear viscosities. The motility enhancements could be explained by the mechanism of stress accumulation in the viscoelastic fluid. This work was supported by the National Science Foundation - DMR-1104705.

  11. Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

    PubMed Central

    Shan, Jiajie; Liao, Jie; Huang, Junwei; Robert, Renaud; Palmer, Melissa L; Fahrenkrug, Scott C; O'Grady, Scott M; Hanrahan, John W

    2012-01-01

    Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl− and HCO3− secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3− net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl− and HCO3− net fluxes. Ieq and HCO3− fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3− is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3−. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3− secretion, suggesting that HCO3− transport under these conditions requires catalysed synthesis of carbonic acid. Cl− was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50–70% of Cl− and fluid transport was bumetanide-insensitive, suggesting basolateral Cl− loading by a sodium–potassium–chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO3− gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO3− secretion was increased by bilateral Cl− removal and therefore did not require apical Cl−/HCO3− exchange. The results suggest a model in which most HCO3− is recycled basolaterally by exchange with Cl−, and the resulting HCO3−-dependent Cl− transport provides an osmotic driving force for

  12. Charging C60 islands with the AFM tip.

    PubMed

    Hoff, Brice; Henry, Claude R; Barth, Clemens

    2016-01-01

    We show that electrons can be transferred on demand from an AFM tip into single bulk-like C60 islands, which are supported on the insulating NaCl(001) surface. We exemplify this by controlled charge-manipulation experiments conducted in ultrahigh vacuum by noncontact AFM (nc-AFM), electrostatic force microscopy (EFM) and Kelvin probe force microscopy (KPFM). KPFM shows a homogeneous contrast at the islands, which is a signature for an equal distribution of the electrons in the T1u band. The charge dissipates during half a day due to an interaction of the charged C60 islands with defects in the near surface region of NaCl. Our results open the perspective in photo-voltaics to study charge attachment, stability and charge exchange with the environment of any C60 bulk-like system. PMID:26617348

  13. Imaging resolution of AFM with probes modified with FIB.

    PubMed

    Skibinski, J; Rebis, J; Wejrzanowski, T; Rozniatowski, K; Pressard, K; Kurzydlowski, K J

    2014-11-01

    This study concerns imaging of the structure of materials using AFM tapping (TM) and phase imaging (PI) mode, using probes modified with focused ion beam (FIB). Three kinds of modifications were applied - thinning of the cantilever, sharpening of the tip and combination of these two modifications. Probes shaped in that way were used for AFM investigations with Bruker AFM Nanoscope 8. As a testing material, titanium roughness standard supplied by Bruker was used. The results show that performed modifications influence the oscillation of the probes. In particular thinning of the cantilever enables one to acquire higher self-resonant frequencies, which can be advantageous for improving the quality of imaging in PI mode. It was found that sharpening the tip improves imaging resolution in tapping mode, which is consistent with existing knowledge, but lowered the quality of high frequency topography images. In this paper the Finite Element Method (FEM) was used to explain the results obtained experimentally. PMID:25080273

  14. AFM of biological complexes: what can we learn?

    PubMed Central

    Gaczynska, Maria; Osmulski, Pawel A.

    2009-01-01

    The term “biological complexes” broadly encompasses particles as diverse as multisubunit enzymes, viral capsids, transport cages, molecular nets, ribosomes, nucleosomes, biological membrane components and amyloids. The complexes represent a broad range of stability and composition. Atomic force microscopy offers a wealth of structural and functional data about such assemblies. For this review, we choose to comment on the significance of AFM to study various aspects of biology of selected nonmembrane protein assemblies. Such particles are large enough to reveal many structural details under the AFM probe. Importantly, the specific advantages of the method allow for gathering dynamic information about their formation, stability or allosteric structural changes critical for their function. Some of them have already found their way to nanomedical or nanotechnological applications. Here we present examples of studies where the AFM provided pioneering information about the biology of complexes, and examples of studies where the simplicity of the method is used toward the development of potential diagnostic applications. PMID:19802337

  15. Improvement in metrology on new 3D-AFM platform

    NASA Astrophysics Data System (ADS)

    Schmitz, Ingo; Osborn, Marc; Hand, Sean; Chen, Qi

    2008-10-01

    According to the 2007 edition of the ITRS roadmap, the requirement for CD uniformity of isolated lines on a binary or attenuated phase shift mask is 2.1nm (3σ) in 2008 and requires improvement to1.3 nm (3σ) in 2010. In order to meet the increasing demand for CD uniformity on photo masks, improved CD metrology is required. A next generation AFM, InSightTM 3DAFM, has been developed to meet these increased requirements for advanced photo mask metrology. The new system achieves 2X improvement in CD and depth precision on advanced photo masks features over the previous generation 3D-AFM. This paper provides measurement data including depth, CD, and sidewall angle metrology. In addition the unique capabilities of damage-free defect inspection and Nanoimprint characterization by 3D AFM are presented.

  16. Cochlear Outer-Hair-Cell Power Generation and Viscous Fluid Loss.

    PubMed

    Wang, Yanli; Steele, Charles R; Puria, Sunil

    2016-01-01

    Since the discovery of otoacoustic emissions and outer hair cell (OHC) motility, the fundamental question of whether the cochlea produces mechanical power remains controversial. In the present work, direct calculations are performed on power loss due to fluid viscosity and power generated by the OHCs. A three-dimensional box model of the mouse cochlea is used with a feed-forward/feed-backward approximation representing the organ of Corti cytoarchitecture. The model is fit to in vivo basilar membrane motion with one free parameter for the OHCs. The calculations predict that the total power output from the three rows of OHCs can be over three orders of magnitude greater than the acoustic input power at 10 dB sound pressure level (SPL). While previous work shows that the power gain, or the negative damping, diminishes with intensity, we show explicitly based on our model that OHC power output increases and saturates with SPL. The total OHC power output is about 2 pW at 80 dB SPL, with a maximum of about 10 fW per OHC. PMID:26792556

  17. Direct visualization of the hydration layer on alumina nanoparticles with the fluid cell STEM in situ

    DOE PAGESBeta

    Firlar, Emre; Çınar, Simge; Kashyap, Sanjay; Akinc, Mufit; Prozorov, Tanya

    2015-05-21

    Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions.more » We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. As a result, our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles.« less

  18. Nitric oxide production by cultured human aortic smooth muscle cells: stimulation by fluid flow

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Tilton, R. G.; Eskin, S. G.; McIntire, L. V.

    1998-01-01

    This study demonstrated that exposure of cultured human aortic smooth muscle cells (SMC) to fluid flow resulted in nitric oxide (NO) production, monitored by nitrite and guanosine 3',5'-cyclic monophosphate production. A rapid burst in nitrite production rate was followed by a more gradual increase throughout the period of flow exposure. Neither the initial burst nor the prolonged nitrite production was dependent on the level of shear stress in the range of 1.1-25 dyn/cm2. Repeated exposure to shear stress after a 30-min static period restimulated nitrite production similar to the initial burst. Ca(2+)-calmodulin antagonists blocked the initial burst in nitrite release. An inhibitor of nitric oxide synthase (NOS) blocked nitrite production, indicating that changes in nitrite reflect NO production. Treatment with dexamethasone or cycloheximide had no effect on nitrite production. Monoclonal antibodies directed against the inducible and endothelial NOS isoforms showed no immunoreactivity on Western blots, whereas monoclonal antibodies directed against the neuronal NOS gave specific products. These findings suggest that human aortic SMC express a constitutive neuronal NOS isoform, the enzymatic activity of which is modulated by flow.

  19. Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ

    PubMed Central

    Thi-Kim Vu, Hanh; Rink, Jochen C; McKinney, Sean A; McClain, Melainia; Lakshmanaperumal, Naharajan; Alexander, Richard; Sánchez Alvarado, Alejandro

    2015-01-01

    Cystic kidney diseases (CKDs) affect millions of people worldwide. The defining pathological features are fluid-filled cysts developing from nephric tubules due to defective flow sensing, cell proliferation and differentiation. The underlying molecular mechanisms, however, remain poorly understood, and the derived excretory systems of established invertebrate models (Caenorhabditis elegans and Drosophila melanogaster) are unsuitable to model CKDs. Systematic structure/function comparisons revealed that the combination of ultrafiltration and flow-associated filtrate modification that is central to CKD etiology is remarkably conserved between the planarian excretory system and the vertebrate nephron. Consistently, both RNA-mediated genetic interference (RNAi) of planarian orthologues of human CKD genes and inhibition of tubule flow led to tubular cystogenesis that share many features with vertebrate CKDs, suggesting deep mechanistic conservation. Our results demonstrate a common evolutionary origin of animal excretory systems and establish planarians as a novel and experimentally accessible invertebrate model for the study of human kidney pathologies. DOI: http://dx.doi.org/10.7554/eLife.07405.001 PMID:26057828

  20. Actuation of flexoelectric membranes in viscoelastic fluids with applications to outer hair cells.

    PubMed

    Herrera-Valencia, E E; Rey, Alejandro D

    2014-11-28

    Liquid crystal flexoelectric actuation uses an imposed electric field to create membrane bending, and it is used by the outer hair cells (OHCs) located in the inner ear, whose role is to amplify sound through generation of mechanical power. Oscillations in the OHC membranes create periodic viscoelastic flows in the contacting fluid media. A key objective of this work on flexoelectric actuation relevant to OHCs is to find the relations and impact of the electromechanical properties of the membrane, the rheological properties of the viscoelastic media, and the frequency response of the generated mechanical power output. The model developed and used in this work is based on the integration of: (i) the flexoelectric membrane shape equation applied to a circular membrane attached to the inner surface of a circular capillary and (ii) the coupled capillary flow of contacting viscoelastic phases, such that the membrane flexoelectric oscillations drive periodic viscoelastic capillary flows, as in OHCs. By applying the Fourier transform formalism to the governing equation, analytical expressions for the transfer function associated with the curvature and electrical field and for the power dissipation of elastic storage energy were found. PMID:25332388

  1. Cerebrospinal fluid-derived Semaphorin3B orients neuroepithelial cell divisions in the apicobasal axis.

    PubMed

    Arbeille, Elise; Reynaud, Florie; Sanyas, Isabelle; Bozon, Muriel; Kindbeiter, Karine; Causeret, Frédéric; Pierani, Alessandra; Falk, Julien; Moret, Frédéric; Castellani, Valérie

    2015-01-01

    The spatial orientation of cell divisions is fundamental for tissue architecture and homeostasis. Here we analysed neuroepithelial progenitors in the developing mouse spinal cord to determine whether extracellular signals orient the mitotic spindle. We report that Semaphorin3B (Sema3B) released from the floor plate and the nascent choroid plexus in the cerebrospinal fluid (CSF) controls progenitor division orientation. Delivery of exogenous Sema3B to neural progenitors after neural tube opening in living embryos promotes planar orientation of their division. Preventing progenitor access to cues present in the CSF by genetically engineered canal obstruction affects the proportion of planar and oblique divisions. Sema3B knockout phenocopies the loss of progenitor access to the CSF. Sema3B binds to the apical surface of mitotic progenitors and exerts its effect via Neuropilin receptors, GSK3 activation and subsequent inhibition of the microtubule stabilizer CRMP2. Thus, extrinsic control mediated by the Semaphorin signalling orients progenitor divisions in neurogenic zones. PMID:25721514

  2. Cochlear Outer-Hair-Cell Power Generation and Viscous Fluid Loss

    PubMed Central

    Wang, Yanli; Steele, Charles R.; Puria, Sunil

    2016-01-01

    Since the discovery of otoacoustic emissions and outer hair cell (OHC) motility, the fundamental question of whether the cochlea produces mechanical power remains controversial. In the present work, direct calculations are performed on power loss due to fluid viscosity and power generated by the OHCs. A three-dimensional box model of the mouse cochlea is used with a feed-forward/feed-backward approximation representing the organ of Corti cytoarchitecture. The model is fit to in vivo basilar membrane motion with one free parameter for the OHCs. The calculations predict that the total power output from the three rows of OHCs can be over three orders of magnitude greater than the acoustic input power at 10 dB sound pressure level (SPL). While previous work shows that the power gain, or the negative damping, diminishes with intensity, we show explicitly based on our model that OHC power output increases and saturates with SPL. The total OHC power output is about 2 pW at 80 dB SPL, with a maximum of about 10 fW per OHC. PMID:26792556

  3. Microvesicles Derived From Human Mesenchymal Stem Cells Restore Alveolar Fluid Clearance in Human Lungs Rejected for Transplantation

    PubMed Central

    Gennai, S.; Monsel, A.; Hao, Q.; Park, J.; Matthay, M. A.; Lee, J. W.

    2016-01-01

    The need to increase the donor pool for lung transplantation is a major public health issue. We previously found that administration of mesenchymal stem cells “rehabilitated” marginal donor lungs rejected for transplantation using ex vivo lung perfusion. However, the use of stem cells has some inherent limitation such as the potential for tumor formation. In the current study, we hypothesized that microvesicles, small anuclear membrane fragments constitutively released from mesenchymal stem cells, may be a good alternative to using stem cells. Using our well established ex vivo lung perfusion model, microvesicles derived from human mesenchymal stem cells increased alveolar fluid clearance (i.e. ability to absorb pulmonary edema fluid) in a dose-dependent manner, decreased lung weight gain following perfusion and ventilation, and improved airway and hemodynamic parameters compared to perfusion alone. Microvesicles derived from normal human lung fibroblasts as a control had no effect. Co-administration of microvesicles with anti-CD44 antibody attenuated these effects, suggesting a key role of the CD44 receptor in the internalization of the microvesicles into the injured host cell and its effect. In summary, microvesicles derived from human mesenchymal stem cells were as effective as the parent mesenchymal stem cells in rehabilitating marginal donor human lungs. PMID:25847030

  4. Charging C60 islands with the AFM tip

    NASA Astrophysics Data System (ADS)

    Hoff, Brice; Henry, Claude R.; Barth, Clemens

    2015-12-01

    We show that electrons can be transferred on demand from an AFM tip into single bulk-like C60 islands, which are supported on the insulating NaCl(001) surface. We exemplify this by controlled charge-manipulation experiments conducted in ultrahigh vacuum by noncontact AFM (nc-AFM), electrostatic force microscopy (EFM) and Kelvin probe force microscopy (KPFM). KPFM shows a homogeneous contrast at the islands, which is a signature for an equal distribution of the electrons in the T1u band. The charge dissipates during half a day due to an interaction of the charged C60 islands with defects in the near surface region of NaCl. Our results open the perspective in photo-voltaics to study charge attachment, stability and charge exchange with the environment of any C60 bulk-like system.We show that electrons can be transferred on demand from an AFM tip into single bulk-like C60 islands, which are supported on the insulating NaCl(001) surface. We exemplify this by controlled charge-manipulation experiments conducted in ultrahigh vacuum by noncontact AFM (nc-AFM), electrostatic force microscopy (EFM) and Kelvin probe force microscopy (KPFM). KPFM shows a homogeneous contrast at the islands, which is a signature for an equal distribution of the electrons in the T1u band. The charge dissipates during half a day due to an interaction of the charged C60 islands with defects in the near surface region of NaCl. Our results open the perspective in photo-voltaics to study charge attachment, stability and charge exchange with the environment of any C60 bulk-like system. Electronic supplementary information (ESI) available. See DOI: 10.1039/C5NR04541J

  5. Molecular signature of amniotic fluid derived stem cells in the fetal sheep model of myelomeningocele.

    PubMed

    Ceccarelli, Gabriele; Pozzo, Enrico; Scorletti, Federico; Benedetti, Laura; Cusella, Gabriella; Ronzoni, Flavio Lorenzo; Sahakyan, Vardine; Zambaiti, Elisa; Mimmi, Maria Chiara; Calcaterra, Valeria; Deprest, Jan; Sampaolesi, Maurilio; Pelizzo, Gloria

    2015-09-01

    Abnormal cord development results in spinal cord damage responsible for myelomeningocele (MMC). Amniotic fluid-derived stem cells (AFSCs) have emerged as a potential candidate for applications in regenerative medicine. However, their differentiation potential is largely unknown as well as the molecular signaling orchestrating the accurate spinal cord development. Fetal lambs underwent surgical creation of neural tube defect and its subsequent repair. AFSCs were isolated, cultured and characterized at the 12th (induction of MMC), 16th (repair of malformation), and 20th week of gestation (delivery). After performing open hysterectomy, AF collections on fetuses with sham procedures at the same time points as the MMC creation group have been used as controls. Cytological analyses with the colony forming unit assay, XTT and alkaline-phosphatase staining, qRT-PCR gene expression analyses (normalized with aged match controls) and NMR metabolomics profiling were performed. Here we show for the first time the metabolomics and molecular signature variation in AFSCs isolated in the sheep model of MMC, which may be used as diagnostic tools for the in utero identification of the neural tube damage. Intriguingly, PAX3 gene involved in the murine model for spina bifida is modulated in AFSCs reaching the peak of expression at 16 weeks of gestation, 4 weeks after the intervention. Our data strongly suggest that AFSCs reorganize their differentiation commitment in order to generate PAX3-expressing progenitors to counteract the MMC induced in the sheep model. The gene expression signature of AFSCs highlights the plasticity of these cells reflecting possible alterations of embryonic development. PMID:26026346

  6. A low-cost AFM setup with an interferometer for undergraduates and secondary-school students

    NASA Astrophysics Data System (ADS)

    Bergmann, Antje; Feigl, Daniela; Kuhn, David; Schaupp, Manuel; Quast, Günter; Busch, Kurt; Eichner, Ludwig; Schumacher, Jens

    2013-07-01

    Atomic force microscopy (AFM) is an important tool in nanotechnology. This method makes it possible to observe nanoscopic surfaces beyond the resolution of light microscopy. In order to provide undergraduate and secondary-school students with insights into this world, we have developed a very robust low-cost AFM setup with a Fabry-Perot interferometer as a detecting device. This setup is designed to be operated almost completely manually and its simplicity gives access to a profound understanding of the working principle. Our AFM is operated in a constant height mode, i.e. the topography of the sample surface is represented directly by the deflection of the cantilever. Thus, the measuring procedure can be understood even by secondary-school students; furthermore, it is the method with the lowest cost, totalling not more than 10-15 k Euros. Nevertheless, we are able to examine a large variety of sample topographies such as CD and DVD surfaces, IC structures, blood cells, butterfly wings or moth eyes. Furthermore, force-distance curves can be recorded and the tensile moduli of some materials can be evaluated. We present our setup in detail and describe its working principles. In addition, we show various experiments which have already been performed by students.

  7. EBNA1 antigen-specific CD8+ T cells in cerebrospinal fluid of patients with multiple sclerosis.

    PubMed

    Erdur, Hebun; Scholz, Veronika; Streitz, Mathias; Hammer, Markus; Meisel, Christian; Schönemann, Constanze; Wandinger, Klaus-Peter; Rosche, Berit

    2016-05-15

    Epidemiological data suggests that Epstein-Barr virus may be involved in the pathogenesis of Multiple Sclerosis (MS). We aimed to determine the frequency of CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) in cerebrospinal fluid (CSF) and blood of patients with MS and other inflammatory neurological diseases (OIND). The frequency of specific CD8+ T cells was assessed by HLA-class-I-binding pentamers restricted to HLA-B35. The frequency of HPVGEADYFEY-specific CD8+ T cells did neither differ significantly in blood nor CSF in MS compared to OIND, but was consistently higher in CSF compared to blood regardless of diagnosis. PMID:27138093

  8. BOREAS AFM-04 Twin Otter Aircraft Flux Data

    NASA Technical Reports Server (NTRS)

    MacPherson, J. Ian; Hall, Forrest G. (Editor); Knapp, David E. (Editor); Desjardins, Raymond L.; Smith, David E. (Technical Monitor)

    2000-01-01

    The BOREAS AFM-5 team collected and processed data from the numerous radiosonde flights during the project. The goals of the AFM-05 team were to provide large-scale definition of the atmosphere by supplementing the existing AES aerological network, both temporally and spatially. This data set includes basic upper-air parameters collected from the network of upper-air stations during the 1993, 1994, and 1996 field campaigns over the entire study region. The data are contained in tabular ASCII files. The data files are available on a CD-ROM (see document number 20010000884) or from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC).

  9. GPIM AF-M315E Propulsion System

    NASA Technical Reports Server (NTRS)

    Spores, Ronald A.; Masse, Robert; Kimbrel, Scott; McLean, Chris

    2014-01-01

    The NASA Space Technology mission Directorate's (STMD) Green Propellant Infusion Mission (GPIM) Technology Demonstration Mission (TDM) will demonstrate an operational AF-M315E green propellant propulsion system. Aerojet-Rocketdyne is responsible for the development of the propulsion system payload. This paper statuses the propulsion system module development, including thruster design and system design; Initial test results for the 1N engineering model thruster are presented. The culmination of this program will be high-performance, green AF-M315E propulsion system technology at TRL 7+, with components demonstrated to TRL 9, ready for direct infusion to a wide range of applications for the space user community.

  10. Evaluation of the Endothelial Cell Antibodies in Serum and Perilymphatic Fluid of Cochlear Implanted Children with Sensorineural Hearing Loss

    PubMed Central

    Farhadi, Mohammad; Noorbakhsh, Samileh; Tabatabaei, Azardokht; Daneshi, Ahamad; Darestani, Sahar Ghavidel; Jomeh, Emam

    2013-01-01

    Introduction Serum Anti endothelial Cell Antibodies (AECAs) play a prominent role in idiopathic Sensorineural Hearing Loss (SNHL) in that they induce vascular damage (immune mediated). The of the current study is To compare AECAs in serum and perilymphatic fluid of idiopathic SNHL children (<15y) undergoing cochlear implant surgery. Methods This was a cross sectional study performed in the cochlear implant ward in Rasoul Akram hospital, Tehran, Iran (2008 -2010) on 99 SNHL children undergoing cochlear implant surgery. The data collected from47 idiopathic and 52 non-idiopathic SNHL cases. AECAs were measured by indirect immuno fluorescence assay and compared in sera and perilymphatic fluids between the two groups. P-value < 0.05 was considered significant. Results Idiopathic SNHL was diagnosed in 47.5% of cases. Positive AECA results in serum and perilymphatic fluid were 10% and 12%, respectively. Although AECA results in perilymphatic fluids were different between idiopathic and non-Idiopathic SNHL patients (PV < 0.05), AECAs in serum showed no significant difference between the two (PV = 0.1). No significant difference was detected between the mean age of idiopathic and non-idiopathic SNHL patients with positive AECAs in serum and perilymphatic fluids (PV = 0.2; PV = 0.2). Discussion Idiopathic SNHL was diagnosed in 47.5% of studied cases. Idiopathic SNHL has a poor out come in children. In cases with idiopathic SNHL, finding AECAs in perilymphatic fluids are more valuable than in the serum. We suggest that serum and perilymphatic fluids testing for AECAs would be helpful in management of idiopathic SNHL cases. Specific immunosuppressive treatments for selected cases suffering from Idiopathic SNHL (only in those older than 5) might be successful in disease management. However, this theory should first be validated by randomized clinical trials. PMID:25337353

  11. A cut-cell finite volume - finite element coupling approach for fluid-structure interaction in compressible flow

    NASA Astrophysics Data System (ADS)

    Pasquariello, Vito; Hammerl, Georg; Örley, Felix; Hickel, Stefan; Danowski, Caroline; Popp, Alexander; Wall, Wolfgang A.; Adams, Nikolaus A.

    2016-02-01

    We present a loosely coupled approach for the solution of fluid-structure interaction problems between a compressible flow and a deformable structure. The method is based on staggered Dirichlet-Neumann partitioning. The interface motion in the Eulerian frame is accounted for by a conservative cut-cell Immersed Boundary method. The present approach enables sub-cell resolution by considering individual cut-elements within a single fluid cell, which guarantees an accurate representation of the time-varying solid interface. The cut-cell procedure inevitably leads to non-matching interfaces, demanding for a special treatment. A Mortar method is chosen in order to obtain a conservative and consistent load transfer. We validate our method by investigating two-dimensional test cases comprising a shock-loaded rigid cylinder and a deformable panel. Moreover, the aeroelastic instability of a thin plate structure is studied with a focus on the prediction of flutter onset. Finally, we propose a three-dimensional fluid-structure interaction test case of a flexible inflated thin shell interacting with a shock wave involving large and complex structural deformations.

  12. Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways.

    PubMed

    Zheng, Lisha; Huang, Yan; Song, Wei; Gong, Xianghui; Liu, Meili; Jia, Xiaolin; Zhou, Gang; Chen, Luoping; Li, Ang; Fan, Yubo

    2012-09-21

    Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12 dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation. PMID:22863019

  13. Cross-omics comparison of stress responses in mesothelial cells exposed to heat- versus filter-sterilized peritoneal dialysis fluids.

    PubMed

    Kratochwill, Klaus; Bender, Thorsten O; Lichtenauer, Anton M; Herzog, Rebecca; Tarantino, Silvia; Bialas, Katarzyna; Jörres, Achim; Aufricht, Christoph

    2015-01-01

    Recent research suggests that cytoprotective responses, such as expression of heat-shock proteins, might be inadequately induced in mesothelial cells by heat-sterilized peritoneal dialysis (PD) fluids. This study compares transcriptome data and multiple protein expression profiles for providing new insight into regulatory mechanisms. Two-dimensional difference gel electrophoresis (2D-DIGE) based proteomics and topic defined gene expression microarray-based transcriptomics techniques were used to evaluate stress responses in human omental peritoneal mesothelial cells in response to heat- or filter-sterilized PD fluids. Data from selected heat-shock proteins were validated by 2D western-blot analysis. Comparison of proteomics and transcriptomics data discriminated differentially regulated protein abundance into groups depending on correlating or noncorrelating transcripts. Inadequate abundance of several heat-shock proteins following exposure to heat-sterilized PD fluids is not reflected on the mRNA level indicating interference beyond transcriptional regulation. For the first time, this study describes evidence for posttranscriptional inadequacy of heat-shock protein expression by heat-sterilized PD fluids as a novel cytotoxic property. Cross-omics technologies introduce a novel way of understanding PDF bioincompatibility and searching for new interventions to reestablish adequate cytoprotective responses. PMID:26495307

  14. The Lineage Specification of Mesenchymal Stem Cells Is Directed by the Rate of Fluid Shear Stress.

    PubMed

    Lu, Juan; Fan, Yijuan; Gong, Xiaoyuan; Zhou, Xin; Yi, Caixia; Zhang, Yinxing; Pan, Jun

    2016-08-01

    The effective regulation of fluid shear stress (FSS) on the lineage specification of mesenchymal stem cells (MSCs) remains to be addressed. We hypothesized that when MSCs are recruited to musculoskeletal system following stimulation, their differentiation into osteogenic or chondrogenic cells is directed by the rate of FSS (ΔSS) through modulation of the mechanosensitive, cation-selective channels (MSCCs), intracellular calcium levels, and F-actin. To this end, MSCs were exposed to laminar FSS linearly increased from 0 to 10 dyn/cm(2) in 0, 2, or 20 min and maintained at 10 dyn/cm(2) for a total of 20 min (termed as ΔSS 0-0', 0-2', and 0-20', respectively, representing more physiological (0-0') and non-physiological (0-2' and 0-20') ΔSS treatments). Our results showed 0-0' facilitated MSC differentiation towards chondrogenic and not osteogenic phenotype, by promoting moderate intracellular calcium concentration ([Ca(2+) ]i ) increase from the calcium channels with the exception of MSCCs or intracellular calcium stores, and F-actin organization. In contrast, 0-2' promoted MSCs towards osteogenic and not chondrogenic phenotype, by inducing significant [Ca(2+) ]i increase mainly from the MSCCs, and F-actin assembly. However, 0-20' elicited the modest osteogenic and chondrogenic phenotypes, as it induced the lowest [Ca(2+) ]i increase mainly from MSCCs, and F-actin assembly. Our results suggest that compared to the more physiological ΔSS, the non-physiological ΔSS favors [Ca(2+) ]i influx from MSCCs. An appropriate non-physiological ΔSS (0-2') even elicits a large [Ca(2+) ]i influx from the MSCCs that reverses the lineage specification of MSCs, providing validation for the high mechanosensitivity of MSCs and guidance for training osteoporosis and osteoarthritis patients. J. Cell. Physiol. 231: 1752-1760, 2016. © 2015 Wiley Periodicals, Inc. PMID:26636289

  15. Response Of Mineralizing And Non-Mineralizing Bone Cells To Fluid Flow: An In Vitro Model For Mechanotransruction

    NASA Technical Reports Server (NTRS)

    Makuch, Lauren A.

    2004-01-01

    Humans reach peak bone mass at age 30. After this point, we lose 1 to 2 percent of bone mass each decade. In the microgravity environment of space, astronauts lose bone mass at an accelerated rate of 1 to 2 percent each month. When astronauts travel to Mars, they may be in space for as long as 3 years. During this time, they may lose about half of their bone mass from weight-bearing bones. This loss may be irreversible. The drastic loss in bone that astronauts experience in space makes them much more vulnerable to fractures. In addition, the corresponding removal of calcium from bone results in higher levels of calcium in the blood, which increases the risk of developing kidney stones. Currently, studies are being conducted which investigate factors governing bone adaptation and mechanotransduction. Bone is constantly adapting in response to mechanical stimuli. Increased mechanical loading stimulates bone formation and suppresses bone resorption. Reduction in mechanical loading caused by bedrest, disuse, or microgravity results in decreased bone formation and possibly increased bone resorption. Osteoblasts and osteoclasts are the two main cell types that participate in bone remodeling. Osteoblasts are anabolic (bone-forming) cells and osteoclasts are catabolic (bone-resorbing) cells. In microgravity, the activity of osteoblasts slows down and the activity of osteoclasts may speed up, causing a loss of bone density. Mechanotransduction, the molecular mechanism by which mechanical stimuli are converted to biochemical signals, is not yet understood. Exposure of cells to fluid flow imposes a shear stress on the cells. Several studies have shown that the shear stress that results from fluid flow induces a cellular response similar to that induced by mechanical loading. Thus, fluid flow can be used as an in vitro model to simulate the mechanical stress that bone cells experience in vivo. Previous in vitro studies have shown that fluid flow induces several responses in

  16. Mesenchymal Stem Cells Obtained from Synovial Fluid Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells on a Matrigel Coating Exhibited Enhanced Proliferation and Differentiation Potential

    PubMed Central

    Zhang, Hong; Liu, Wen-Jing; Jiang, Rui; Li, Wen-Yu; Zheng, You-Hua; Zhang, Zhi-Guang

    2015-01-01

    Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) serve as a promising source for cell-based therapies in regenerative medicine. However, optimal methods for transforming iPSCs into MSCs and the characteristics of iPSC-MSCs obtained from different methods remain poorly understood. In this study, we developed a one-step method for obtaining iPSC-MSCs (CD146+STRO-1+ MSCs) from human synovial fluid MSC-derived induced iPSCs (SFMSC-iPSCs). CD146-STRO-1-SFMSCs were reprogrammed into iPSCs by transduction with lentivirus-mediated Sox2, Oct-3/4, klf4, and c-Myc. SFMSC-iPSCs were maintained with mTeSR1 medium in Matrigel-coated culture plates. Single dissociated cells were obtained by digesting the SFMSC-iPSCs with trypsin. The dissociated cells were then plated into Matrigel-coated culture plate with alpha minimum essential medium supplemented with 10% fetal bovine serum, 1× Glutamax, and the ROCK inhibitor Y-27632. Cells were then passaged in standard cell culture plates with alpha minimum essential medium supplemented with 10% fetal bovine serum and 1× Glutamax. After passaging in vitro, the cells showed a homogenous spindle-shape similar to their ancestor cells (SFMSCs), but with more robust proliferative activity. Flow cytometric analysis revealed typical MSC surface markers, including expression of CD73, CD90, CD105, and CD44 and lack of CD45, CD34, CD11b, CD19, and HLA-DR. However, these cells were positive for CD146 and stro-1, which the ancestor cells were not. Moreover, the cells could also be induced to differentiate in osteogenic, chondrogenic, and adipogenic lineages in vitro. The differentiation potential was improved compared with the ancestor cells in vitro. The cells were not found to exhibit oncogenicity in vivo. Therefore, the method presented herein facilitated the generation of STRO-1+CD146+ MSCs from SFMSC-iPSCs exhibiting enhanced proliferation and differentiation potential. PMID:26649753

  17. AFM CHARACTERIZATION OF LASER INDUCED DAMAGE ON CDZNTE CRYSTAL SURFACES

    SciTech Connect

    Hawkins, S; Lucile Teague, L; Martine Duff, M; Eliel Villa-Aleman, E

    2008-06-10

    Semi-conducting CdZnTe (or CZT) crystals can be used in a variety of detector-type applications. CZT shows great promise for use as a gamma radiation spectrometer. However, its performance is adversely affected by point defects, structural and compositional heterogeneities within the crystals, such as twinning, pipes, grain boundaries (polycrystallinity), secondary phases and in some cases, damage caused by external forces. One example is damage that occurs during characterization of the surface by a laser during Raman spectroscopy. Even minimal laser power can cause Te enriched areas on the surface to appear. The Raman spectra resulting from measurements at moderate intensity laser power show large increases in peak intensity that is attributed to Te. Atomic Force Microscopy (AFM) was used to characterize the extent of damage to the CZT crystal surface following exposure to the Raman laser. AFM data reveal localized surface damage in the areas exposed to the Raman laser beam. The degree of surface damage to the crystal is dependent on the laser power, with the most observable damage occurring at high laser power. Moreover, intensity increases in the Te peaks of the Raman spectra are observed even at low laser power with little to no visible damage observed by AFM. AFM results also suggest that exposure to the same amount of laser power yields different amounts of surface damage depending on whether the exposed surface is the Te terminating face or the Cd terminating face of CZT.

  18. 3D Color Digital Elevation Map of AFM Sample

    NASA Technical Reports Server (NTRS)

    2008-01-01

    This color image is a three dimensional (3D) view of a digital elevation map of a sample collected by NASA's Phoenix Mars Lander's Atomic Force Microscope (AFM).

    The image shows four round pits, only 5 microns in depth, that were micromachined into the silicon substrate, which is the background plane shown in red. This image has been processed to reflect the levelness of the substrate.

    A Martian particle only one micrometer, or one millionth of a meter, across is held in the upper left pit.

    The rounded particle shown at the highest magnification ever seen from another world is a particle of the dust that cloaks Mars. Such dust particles color the Martian sky pink, feed storms that regularly envelop the planet and produce Mars' distinctive red soil.

    The particle was part of a sample informally called 'Sorceress' delivered to the AFM on the 38th Martian day, or sol, of the mission (July 2, 2008). The AFM is part of Phoenix's microscopic station called MECA, or the Microscopy, Electrochemistry, and Conductivity Analyzer.

    The AFM was developed by a Swiss-led consortium, with Imperial College London producing the silicon substrate that holds sampled particles.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  19. Structural investigations on native collagen type I fibrils using AFM

    SciTech Connect

    Strasser, Stefan; Zink, Albert; Janko, Marek; Heckl, Wolfgang M.; Thalhammer, Stefan . E-mail: stefan.thalhammer@gsf.de

    2007-03-02

    This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.

  20. Hydrodynamic effects in fast AFM single-molecule force measurements.

    PubMed

    Janovjak, Harald; Struckmeier, Jens; Müller, Daniel J

    2005-02-01

    Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor-ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few microm/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 microm/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 microm/s pulling speed. PMID:15257425

  1. AFM Structural Characterization of Drinking Water Biofilm under Physiological Conditions

    EPA Science Inventory

    Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air...

  2. Conductive supports for combined AFM SECM on biological membranes

    NASA Astrophysics Data System (ADS)

    Frederix, Patrick L. T. M.; Bosshart, Patrick D.; Akiyama, Terunobu; Chami, Mohamed; Gullo, Maurizio R.; Blackstock, Jason J.; Dooleweerdt, Karin; de Rooij, Nico F.; Staufer, Urs; Engel, Andreas

    2008-09-01

    Four different conductive supports are analysed regarding their suitability for combined atomic force and scanning electrochemical microscopy (AFM-SECM) on biological membranes. Highly oriented pyrolytic graphite (HOPG), MoS2, template stripped gold, and template stripped platinum are compared as supports for high resolution imaging of reconstituted membrane proteins or native membranes, and as electrodes for transferring electrons from or to a redox molecule. We demonstrate that high resolution topographs of the bacterial outer membrane protein F can be recorded by contact mode AFM on all four supports. Electrochemical feedback experiments with conductive cantilevers that feature nanometre-scale electrodes showed fast re-oxidation of the redox couple Ru(NH3)63+/2+ with the two metal supports after prolonged immersion in electrolyte. In contrast, the re-oxidation rates decayed quickly to unpractical levels with HOPG or MoS2 under physiological conditions. On HOPG we observed heterogeneity in the re-oxidation rate of the redox molecules with higher feedback currents at step edges. The latter results demonstrate the capability of conductive cantilevers with small electrodes to measure minor variations in an SECM signal and to relate them to nanometre-scale features in a simultaneously recorded AFM topography. Rapid decay of re-oxidation rate and surface heterogeneity make HOPG or MoS2 less attractive for combined AFM-SECM experiments on biological membranes than template stripped gold or platinum supports.

  3. Scaling crossover in thin-film drag dynamics of fluid drops in the Hele-Shaw cell

    PubMed Central

    Yahashi, Misato; Kimoto, Natsuki; Okumura, Ko

    2016-01-01

    We study both experimentally and theoretically the descending motion due to gravity of a fluid drop surrounded by another immiscible fluid in a confined space between two parallel plates, i.e., in the Hele-Shaw cell. As a result, we show a new scaling regime of a nonlinear drag friction in viscous liquid that replaces the well-known Stokes’ drag friction through a clear collapse of experimental data thanks to the scaling law. In the novel regime, the dissipation in the liquid thin film formed between the drop and cell walls governs the dynamics. The crossover of this scaling regime to another scaling regime in which the dissipation inside the droplet is dominant is clearly demonstrated and a phase diagram separating these scaling regimes is presented. PMID:27562151

  4. Scaling crossover in thin-film drag dynamics of fluid drops in the Hele-Shaw cell.

    PubMed

    Yahashi, Misato; Kimoto, Natsuki; Okumura, Ko

    2016-01-01

    We study both experimentally and theoretically the descending motion due to gravity of a fluid drop surrounded by another immiscible fluid in a confined space between two parallel plates, i.e., in the Hele-Shaw cell. As a result, we show a new scaling regime of a nonlinear drag friction in viscous liquid that replaces the well-known Stokes' drag friction through a clear collapse of experimental data thanks to the scaling law. In the novel regime, the dissipation in the liquid thin film formed between the drop and cell walls governs the dynamics. The crossover of this scaling regime to another scaling regime in which the dissipation inside the droplet is dominant is clearly demonstrated and a phase diagram separating these scaling regimes is presented. PMID:27562151

  5. Charge Content In Nanometer Rings from Atomic Force Microscope (AFM) Traces

    NASA Astrophysics Data System (ADS)

    Zypman, F.; Eppell, S.; Feinstein, M.; Fried, Y.; Lazarev, D.; Metzger, C.

    The last few years have seen a growing interest in identifying charge content in small structures such as graphene ribbons and aromatic biorings. More generally it is believed that charge content in proteins holds the key to the ultimate understanding of biological self-assembly. Here we describe a model system, a charged ring inside liquid probed by an AFM tip, and show how the charge content and the relative size of the ring with respect to the tip affect the measured force. More importantly, we explain how to measure the charge from the AFM experimental data. The process involves the modeling of the dynamics of the tip-cantilever sensor under the influence of the charged sample, but also of ambient hydrodynamic forces, electrostatic interactions that appear due to charge induction in the tip and electrolytic screening. Of particular relevance is the possibility of our approach to treat analytically the size of ions. This is relevant when the tip-sample distance becomes sub-nanometric, and the more common description via Poisson-Boltzmann equation breaks down. Funding for this research ``Instrument Development: Charge Sensing In Fluids With Nanometer Precision'' is provided by Chemical Measurement & Imaging, National Science Foundation, Grant Number 1508085.

  6. XPS and AFM analysis of antifouling PEG interfaces for microfabricated silicon biosensors.

    PubMed

    Sharma, Sadhana; Johnson, Robert W; Desai, Tejal A

    2004-09-15

    In the past two decades, the biological and medical fields have seen great advances in the development of biosensors capable of quantifying biomolecules. Many of these biosensors have micro- and nano-scale features, are fabricated using biochip technology, and use silicon as a base material. The creation of antifouling sensor interfaces is critical to avoid serious consequences that arise due to their contact with biological fluids. To this end, we have created thin PEG interfaces of various grafting densities on silicon using a single-step PEG-silane coupling reaction scheme. Initial PEG concentration (5-50 mM) and coupling time (0.5-24 h) were varied to attain different grafting densities, and different PEG interfaces so created were analyzed using XPS and AFM. Furthermore, all the PEG interfaces were evaluated using XPS and AFM for their antifouling abilities using fibrinogen as the model protein. Results indicated that PEG interfaces created in this investigation are appropriate for biosensors with micro- and nano-scale features, and are efficient in controlling protein fouling. PMID:15308226

  7. Fast image scanning method in liquid-AFM without image distortion.

    PubMed

    Choi, Inhee; Kim, Younghun; Kim, Jong Ho; Yang, Young In; Lee, Jeongjin; Lee, Suseung; Hong, Surin; Yi, Jongheop

    2008-11-01

    High speed imaging by atomic force microscopy (AFM) allows one to directly observe the dynamic behavior of a sample surface immersed in liquid media; thus, it has been considered to be an indispensable tool for nanobiotechnology and is used in many research fields, including molecular biology and surface science. For real-time observation of a certain behavior, the high speed imaging technique should be accompanied with a high resolution imaging technique to identify target materials. To improve the image quality at a high scanning rate, we developed a variable-controlled fast scanning method, which originated from the modified squeeze-drag superposition model in liquid media. A collection of non-distorted images was accomplished after proper modification of the operating conditions in a viscous fluid, via the simple handling of loading force and cantilever length. Consequently, a speeded-up AFM imaging process was achieved in the liquid environment at up to 200 µm s(-1), without attachment of additional devices. The reliability of the proposed method was verified by the characterization of a grating sample immersed in three types of liquid media. In addition, the results were visualized for elastic biomolecules submerged in a liquid with high kinematic viscosity. PMID:21832743

  8. Insulated Conducting Cantilevered Nanotips and Two-Chamber Recording System for High Resolution Ion Sensing AFM

    PubMed Central

    Meckes, Brian; Arce, Fernando Teran; Connelly, Laura S.; Lal, Ratnesh

    2014-01-01

    Biological membranes contain ion channels, which are nanoscale pores allowing controlled ionic transport and mediating key biological functions underlying normal/abnormal living. Synthetic membranes with defined pores are being developed to control various processes, including filtration of pollutants, charge transport for energy storage, and separation of fluids and molecules. Although ionic transport (currents) can be measured with single channel resolution, imaging their structure and ionic currents simultaneously is difficult. Atomic force microscopy enables high resolution imaging of nanoscale structures and can be modified to measure ionic currents simultaneously. Moreover, the ionic currents can also be used to image structures. A simple method for fabricating conducting AFM cantilevers to image pore structures at high resolution is reported. Tungsten microwires with nanoscale tips are insulated except at the apex. This allows simultaneous imaging via cantilever deflections in normal AFM force feedback mode as well as measuring localized ionic currents. These novel probes measure ionic currents as small as picoampere while providing nanoscale spatial resolution surface topography and is suitable for measuring ionic currents and conductance of biological ion channels. PMID:24663394

  9. HTLV-I associated arthritis: characteristics of an HTLV-I virus infected T cell line from synovial fluid.

    PubMed Central

    Eguchi, K; Nakamura, T; Mine, M; Ida, H; Kawakami, A; Migita, K; Nagasato, K; Kurata, A; Fukuda, T; Nagataki, S

    1992-01-01

    A T cell line from mononuclear cells in the synovial fluid of a patient with polyarthritis was established. The T cell line reacted with serum samples positive for antibodies to human T cell lymphotropic virus type I (HTLV-I) and with monoclonal antibody to HTLV-I p19. In Southern blotting with an env-pX-LTR HTLV-I probe and digestion of T cell line DNA with the restriction enzymes ClaI, DraI, and PstI generated fragments that were identical to those found in two HTLV-I infected T cell lines established from adult T cell leukaemia or HTLV-I associated myelopathy. The T cell line expressed CD2, CD3, CD4, CD45RA, CD29, HLA-DR, CD25, and CD26 antigens, but not CD8 and CD20 antigens. Large amounts of interleukin 6, interferon gamma, and tumour necrosis factor alpha were secreted in the culture supernatants of this cell line. This line helped immunoglobulin production by B cells, but not K562, Raji, and synovial cell lysis. Images PMID:1616338

  10. Periodontal Treatment Downregulates Protease-Activated Receptor 2 in Human Gingival Crevicular Fluid Cells

    PubMed Central

    Euzebio Alves, Vanessa Tubero; Bueno da Silva, Henrique Aparecido; de França, Bruno Nunes; Eichler, Rosangela Santos; Saraiva, Luciana; de Carvalho, Maria Helena Catelli

    2013-01-01

    Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation. PMID:24042113

  11. Comparison of human mesenchymal stem cells derived from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous tooth pulp.

    PubMed

    Isobe, Y; Koyama, N; Nakao, K; Osawa, K; Ikeno, M; Yamanaka, S; Okubo, Y; Fujimura, K; Bessho, K

    2016-01-01

    Populations of pluripotent stem cells were isolated from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous teeth and their multipotentiality properties compared. Osteogenic, chondrogenic, adipogenic, and neurogenic differentiation potentials were examined. Bone marrow mesenchymal stem cells (BMMSCs) and synovial fluid-derived cells (SFCs) showed the highest levels of osteogenesis as expressed by alkaline phosphatase (ALP) activity (0.54±0.094 U/mg protein and 0.57±0.039 U/mg protein, respectively; P=0.60) and by osteocalcin (BGLAP; determined by real-time RT-PCR). SFCs showed the highest levels of chondrogenesis as expressed by ALP activity (1.75±0.097 U/mg protein) and of COL2A1 and COL10A1 by real-time PCR. In terms of adipogenesis, lipid vesicles were observed in the BMMSCs and SFCs. Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) exhibited neurogenesis potential, as shown by increases in expression of class III β-tubulin (TUBB3) and microtubule-associated protein 2 (MAP2) on RT-PCR. Variability was found in the differentiation potential corresponding to the tendency of the original tissue to differentiate. It is suggested that the cell type should be selected depending on the regenerative treatment regimen. PMID:26235629

  12. Production of Bovine Embryos and Calves Cloned by Nuclear Transfer Using Mesenchymal Stem Cells from Amniotic Fluid and Adipose Tissue.

    PubMed

    da Silva, Carolina Gonzales; Martins, Carlos Frederico; Cardoso, Tereza Cristina; da Cunha, Elisa Ribeiro; Bessler, Heidi Christina; Martins, George Henrique Lima; Pivato, Ivo; Báo, Sônia Nair

    2016-04-01

    The less differentiated the donor cells are used in nuclear transfer (NT), the more easily are they reprogrammed by the recipient cytoplasm. In this context, mesenchymal stem cells (MSCs) appear as an alternative to donor nuclei for NT. The amniotic fluid and adipose tissue are sources of MSCs that have not been tested for the production of cloned embryos in cattle. The objective of this study was to isolate, characterize, and use MSCs derived from amniotic fluid (MSC-AF) and adipose tissue (MSC-AT) to produce cloned calves. Isolation of MSC-AF was performed using in vivo ultrasound-guided transvaginal amniocentesis, and MSC-AT were isolated by explant culture. Cellular phenotypic and genotypic characterization by flow cytometry, immunohistochemistry, and RT-PCR were performed, as well as induction in different cell lineages. The NT was performed using MSC-AF and MSC-AT as nuclear donors. The mesenchymal markers of MSC were expressed in bovine MSC-AF and MSC-AT cultures, as evidenced by flow cytometry, immunohistochemistry, and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes, and adipocytes. Embryo production was similar between the cell types, and two calves were born. The calf from MSC-AT was born healthy, and this fact opens a new possibility of using this type of cell to produce cloned cattle by NT. PMID:27055630

  13. Novel Polymer Linkers for Single Molecule AFM Force Spectroscopy

    PubMed Central

    Tong, Zenghan; Mikheikin, Andrey; Krasnoslobodtsev, Alexey; Lv, Zhengjian; Lyubchenko, Yuri L.

    2013-01-01

    Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aβ42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications. PMID:23624104

  14. MOS-based nanocapacitor using C-AFM

    NASA Astrophysics Data System (ADS)

    Hill, Daniel; Sadewasser, Sascha; Aymerich, Xavier

    2003-04-01

    This report details the attempts made to realise nanocapacitors for nanoscale MOS based integrated circuits by AFM anodic oxidation, and therefore isolation, of nano-sized squares of poly-silicon, titanium and aluminium on Si/SiO2. Conductive AFM (C-AFM) was used to perform topographical and electrical characterisation. The experiments were performed with contact mode C-AFM, in ambient air, using Pt-Ir, Co-Cr and Ti coated (20nm) n-type silicon cantilevers. Each sample consisted of a 3-5nm thick conductor deposited on 6nm of SiO2, which was thermally grown on Phosphorus doped (1019 cm-3) n-type Si(100) substrates. Standard cleaning and passivation processes were used. Poly-silicon was immediately found to be too rough to oxidise. Initial current-voltage measurements inside of the titanium-oxide squares suggest initial isolation followed by degradation through Fowler-Nordheim tunnelling. Measurement inconsistencies seen suggest charge storage on the surface or tip with the barrier height of the native titanium oxide thought to be responsible. Al has a thicker natural oxide. To overcome this we designed a series of structures consisting of a Ti finger on SiO2, that is connected to a Ti bond pad, allowing direct probing by a semiconductor parameter analyser. AFM anodic oxidation was performed upon these Ti fingers to reduce their in-plane dimensions towards the nanoscale. To confirm the existence of a nanocapacitor topographical and electrical measurements were then done on and around them.

  15. Single Dimer E-Cadherin Interaction Forces Characterized Using Modified AFM Cantilevers

    NASA Astrophysics Data System (ADS)

    Rudnitsky, Robert; Drees, Frauke; Nelson, W. James; Kenny, Thomas

    2002-03-01

    In tissue monolayers, adhesion between cells is accomplished chiefly through the action of [Ca++] dependent cadherin proteins. E-cadherin molecules coalesce into large plaques on contacting membranes of adjacent cells. Using specialized AFM cantilevers functionalized with tethered E-cadherin proteins, we studied the interaction forces of trans dimers from the single bond level through to the higher surface densities found in plaques, with pico-Newton force resolution. The measurements demonstrated the dependence of E-cadherin homoassociation on surface protein density. Previous in-vivo studies established the role of Ca++ in E-cadherin adhesion in whole cells. Advances in AFM force spectroscopy allowed us to characterize the unbinding process under force loads, and to differentiate single and multiple molecular binding events. The data correlates the dependence of E-cadherin adhesion at a molecular level to [Ca++], revealing interaction details that cannot be observed using whole-cell studies. This work is supported by NSF (XYZ on a Chip Program) CMS-9980838, NIH (GMB5227), and the Fannie and John Hertz Foundation.

  16. Comparison of the Identation and Elasticity of E.coli and its Spheroplasts by AFM

    SciTech Connect

    Sullivan, Claretta J; Venkataraman, Sankar; Retterer, Scott T; Allison, David P; Doktycz, Mitchel John

    2007-01-01

    Atomic force microscopy (AFM) provides a unique opportunity to study live individual bacteria at the nanometer scale. In addition to providing accurate morphological information, AFM can be exploited to investigate membrane protein localization and molecular interactions on the surface of living cells. A prerequisite for these studies is the development of robust procedures for sample preparation. While such procedures are established for intact bacteria, they are only beginning to emerge for bacterial spheroplasts. Spheroplasts are useful research models for studying mechanosensitive ion channels, membrane transport, lipopolysaccharide translocation, solute uptake, and the effects of antimicrobial agents on membranes. Furthermore, given the similarities between spheroplasts and cell wall-deficient (CWD) forms of pathogenic bacteria, spheroplast research could be relevant in biomedical research. In this paper, a new technique for immobilizing spheroplasts on mica pretreated with aminopropyltriethoxysilane (APTES) and glutaraldehyde is described. Using this mounting technique, the indentation and cell elasticity of glutaraldehyde-fixed and untreated spheroplasts of E. coli in liquid were measured. These values are compared to those of intact E. coli. Untreated spheroplasts were found to be much softer than the intact cells and the silicon nitride cantilevers used in this study.

  17. Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways.

    PubMed

    Govey, Peter M; Jacobs, Jon M; Tilton, Susan C; Loiselle, Alayna E; Zhang, Yue; Freeman, Willard M; Waters, Katrina M; Karin, Norman J; Donahue, Henry J

    2014-06-01

    Osteocytes, positioned within bone׳s porous structure, are subject to interstitial fluid flow upon whole bone loading. Such fluid flow is widely theorized to be a mechanical signal transduced by osteocytes, initiating a poorly understood cascade of signaling events mediating bone adaptation to mechanical load. The objective of this study was to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels using high-throughput approaches. Osteocyte-like MLO-Y4 cells were subjected to 2h of oscillating fluid flow (1Pa peak shear stress) and analyzed following 0, 2, 8, and 24h post-flow incubation. Transcriptomic microarray analysis, followed by gene ontology pathway analysis, demonstrated fluid flow regulation of genes consistent with both known and unknown metabolic and inflammatory responses in bone. Additionally, two of the more highly up-regulated gene products - chemokines Cxcl1 and Cxcl2, supported by qPCR - have not previously been reported as responsive to fluid flow. Proteomic analysis demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. Finally, an integrative pathway analysis merging fold changes in transcript and protein levels predicted signaling nodes not directly detected at the sampled time points, including transcription factors c-Myc, c-Jun, and RelA/NF-κB. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as possible paracrine agents for osteoblastic and osteoclastic recruitment. Moreover, these results demonstrate the utility of integrative, high-throughput approaches in place of a traditional candidate approach for identifying novel mechano-sensitive signaling molecules. PMID:24720889

  18. Integrative Transcriptomic and Proteomic Analysis of Osteocytic Cells Exposed to Fluid Flow Reveals Novel Mechano-Sensitive Signaling Pathways

    PubMed Central

    Govey, Peter M.; Jacobs, Jon M.; Tilton, Susan C.; Loiselle, Alayna E.; Zhang, Yue; Freeman, Willard M.; Waters, Katrina M.; Karin, Norman J.; Donahue, Henry J.

    2014-01-01

    Osteocytes, positioned within bone's porous structure, are subject to interstitial fluid flow upon whole bone loading. Such fluid flow is widely theorized to be a mechanical signal transduced by osteocytes, initiating a poorly understood cascade of signaling events mediating bone adaptation to mechanical load. The objective of this study was to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels using high-throughput approaches. Osteocyte-like MLO-Y4 cells were subjected to 2 hours of oscillating fluid flow (1 Pa peak shear stress) and analyzed following 0, 2, 8, and 24 hours post-flow incubation. Transcriptomic microarray analysis, followed by gene ontology pathway analysis, demonstrated fluid flow regulation of genes consistent with both known and unknown metabolic and inflammatory responses in bone. Additionally, two of the more highly up-regulated gene products—chemokines Cxcl1 and Cxcl2, supported by qPCR—have not previously been reported as responsive to fluid flow. Proteomic analysis demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. Finally, an integrative pathway analysis merging fold changes in transcript and protein levels predicted signaling nodes not directly detected at the sampled time points, including transcription factors c-Myc, c-Jun, and RelA/NF-κB. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as possible paracrine agents for osteoblastic and osteoclastic recruitment. Moreover, these results demonstrate the utility of integrative, high-throughput approaches in place of a traditional candidate approach for identifying novel mechano-sensitive signaling molecules. PMID:24720889

  19. A dynamic pressure view cell for acoustic stimulation of fluids—Micro-bubble generation and fluid movement in porous media

    NASA Astrophysics Data System (ADS)

    Stewart, Robert A.; Shaw, J. M.

    2015-09-01

    The development and baseline operation of an acoustic view cell for observing fluids, and fluid-fluid and fluid-solid interfaces in porous media over the frequency range of 10-5000 Hz is described. This range includes the industrially relevant frequency range 500-5000 Hz that is not covered by existing devices. Pressure waveforms of arbitrary shape are generated in a 17.46 mm ID by 200 mm and 690.5 mm long glass tubes at flow rates up to 200 ml/min using a syringe pump. Peak-to-peak amplitudes exceeding 80 kPa are readily realized at frequencies from 10 to 5000 Hz in bubble free fluids when actuated with 20 Vpp as exemplified using castor oil. At resonant frequencies, peak-to-peak pressure amplitudes exceeding 500 kPa were obtained (castor oil at 2100 Hz when actuated with 20 Vpp). Impacts of vibration on macroscopic liquid-liquid and liquid-vapour interfaces and interface movement are illustrated. Pressure wave transmission and attenuation in a fluid saturated porous medium, randomly packed 250-330 μm spherical silica beads, is also demonstrated. Attenuation differences and frequency shifts in resonant peaks are used to detect the presence and generation of dispersed micro-bubbles (<180 μm diameter), and bubbles within porous media that are not readily visualized. Envisioned applications include assessment of the impacts of vibration on reaction, mass transfer, and flow/flow pattern outcomes. This knowledge will inform laboratory and pilot scale process studies, where nuisance vibrations may affect the interpretation of process outcomes, and large scale or in situ processes in aquifers or hydrocarbon reservoirs where imposed vibration may be deployed to improve aspects of process performance. Future work will include miscible interface observation and quantitative measurements in the bulk and in porous media where the roles of micro-bubbles comprise subjects of special interest.

  20. Resveratrol Protects Chondrocytes from Apoptosis via Altering the Ultrastructural and Biomechanical Properties: An AFM Study

    PubMed Central

    Chen, Tongsheng; Wang, Xiaoping

    2014-01-01

    Osteoarthritis (OA), a degenerative joint disease with high prevalence among older people, occurs from molecular or nanometer level and extends gradually to higher degrees of the ultrastructure of cartilage, finally resulting in irreversible structural and functional damages. This report aims to use atomic force microscopy (AFM) to investigate the protective effects of resveratrol (RV), a drug with good anti-inflammatory properties, on cellular morphology, membrane architecture, cytoskeleton, cell surface adhesion and stiffness at nanometer level in sodium nitroprusside (SNP)-induced apoptotic chondrocytes, a typical cellular OA model. CCK-8 assay showed that 100 μM RV significantly prevented SNP-induced cytotoxicity. AFM imaging and quantitative analysis showed that SNP potently induced chondrocytes changes including shrunk, round, lamellipodia contraction and decrease in adherent junctions among cells, as well as the destruction of biomechanics: 90% decrease in elasticity and 30% decrease in adhesion. In addition, confocal imaging analysis showed that SNP induced aggregation of the cytoskeleton and decrease in the expression of cytoskeletal proteins. More importantly, these SNP-induced damages to chondrocytes could be potently prevented by RV pretreatment. Interestingly, the biomechanical changes occurred before morphological changes could be clearly observed during SNP-induced apoptosis, indicating that the biomechanics of cellular membrane may be a more robust indicator of cell function. Collectively, our data demonstrate that RV prevents SNP-induced apoptosis of chondrocytes by regulating actin organization, and that AFM-based technology can be developed into a powerful and sensitive method to study the interaction mechanisms between chondrocytes and drugs. PMID:24632762

  1. Proteomic analysis of human osteoarthritis synovial fluid

    PubMed Central

    2014-01-01

    Background Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography. Results We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples. Conclusions We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the

  2. Sorting of membrane and fluid at the apical pole of polarized Madin-Darby canine kidney cells.

    PubMed

    Leung, S M; Ruiz, W G; Apodaca, G

    2000-06-01

    When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (fluid-rich apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome. PMID:10848634

  3. Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

    PubMed

    Krimmel, Jeffrey D; Schmitt, Michael W; Harrell, Maria I; Agnew, Kathy J; Kennedy, Scott R; Emond, Mary J; Loeb, Lawrence A; Swisher, Elizabeth M; Risques, Rosa Ana

    2016-05-24

    Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue. PMID:27152024

  4. Sorting of Membrane and Fluid at the Apical Pole of Polarized Madin-Darby Canine Kidney Cells

    PubMed Central

    Leung, Som-Ming; Ruiz, Wily G.; Apodaca, Gerard

    2000-01-01

    When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (≤2.5 min at 37°C) and are then rapidly segregated from one another. The dextran remains in the large supranuclear EEA1-positive early endosomes while recycling polymeric immunoglobulin receptor–bound immunoglobulin A is delivered to a Rab11-positive subapical recycling compartment. This latter step requires an intact microtubule cytoskeleton. Receptor-bound transferrin, a marker of the basolateral recycling pathway, has limited access to the fluid-rich apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome. PMID:10848634

  5. Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells

    NASA Technical Reports Server (NTRS)

    McAllister, T. N.; Du, T.; Frangos, J. A.

    2000-01-01

    Bone is a porous tissue that is continuously perfused by interstitial fluid. Fluid flow, driven by both vascular pressure and mechanical loading, may generate significant shear stresses through the canaliculi as well as along the bone lining at the endosteal surface. Both osteoblasts and osteocytes produce signaling factors such as prostaglandins and nitric in response to fluid shear stress (FSS); however, these humoral agents appear to have more profound affects on osteoclast activity at the endosteal surface. We hypothesized that osteoclasts and preosteoclasts may also be mechanosensitive and that osteoclast-mediated autocrine signaling may be important in bone remodeling. In this study, we investigated the effect of FSS on nitric oxide (NO), prostaglandin E(2) (PGE(2)), and prostacyclin (PGI(2)) release by neonatal rat bone marrow-derived preosteoclast-like cells. These cells were tartrate-resistant acid phosphatase (TRAP) positive, weakly nonspecific esterase (NSE) positive, and capable of fusing into calcitonin-responsive, bone-resorbing, multinucleated cells. Bone marrow-derived preosteoclast-like cells exposed for 6 h to a well-defined FSS of 16 dynes/cm(2) produced NO at a rate of 7.5 nmol/mg protein/h, which was 10-fold that of static controls. This response was completely abolished by 100 microM N(G)-amino-L-arginine (L-NAA). Flow also stimulated PGE(2) production (3.9 microg/mg protein/h) and PGI(2) production (220 pg/mg protein/h). L-NAA attenuated flow-induced PGE(2) production by 30%, suggesting that NO may partially modulate PGE(2) production. This is the first report demonstrating that marrow derived cells are sensitive to FSS and that autocrine signaling in these cells may play an important role in load-induced remodeling and signal transduction in bone. Copyright 2000 Academic Press.

  6. Cerebroside Sulfatase Activity in Cultivated Human Skin Fibroblasts and Amniotic Fluid Cells

    ERIC Educational Resources Information Center

    Booth, Carol W.; And Others

    1975-01-01

    Prenatal monitoring for metachromatic leukodystrophy (a fatal inherited metabolic disorder) suggested that the determination of levels of cerebroside sulfatase in the amniotic fluid helped in the prenatal detection of this disorder. (DB)

  7. Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

    PubMed

    Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

    2012-03-01

    Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products. PMID:22090422

  8. Monitoring Cellular Events in Living Mast Cells Stimulated with an Extremely Small Amount of Fluid on a Microchip

    NASA Astrophysics Data System (ADS)

    Munaka, Tatsuya; Abe, Hirohisa; Kanai, Masaki; Sakamoto, Takashi; Nakanishi, Hiroaki; Yamaoka, Tetsuji; Shoji, Shuichi; Murakami, Akira

    2006-07-01

    We successfully developed a measurement system for real-time analysis of cellular function using a newly designed microchip. This microchip was equipped with a micro cell incubation chamber (240 nl) and was stimulated by a very small amount of stimuli (as small as 24 nl). Using the microchip system, cultivation of mast cells was successfully carried out. Monitoring of the cellular events after stimulation with an extremely small amount of fluid on a microchip was performed. This system could be applicable for various types of cellular analysis including real-time monitoring of cellular response by stimulation.

  9. Intramuscular Transplantation of Pig Amniotic Fluid-Derived Progenitor Cells Has Therapeutic Potential in a Mouse Model of Myocardial Infarction.

    PubMed

    Peng, Shao-Yu; Chou, Chih-Jen; Cheng, Po-Jen; Tseng, Tse-Yang; Cheng, Winston Teng-Kui; Shaw, S W Steven; Wu, Shinn-Chih

    2015-01-01

    Acute myocardial infarction (MI) is a fatal event that causes a large number of deaths worldwide. MI results in pathological remodeling and decreased cardiac function, which could lead to heart failure and fatal arrhythmia. Cell therapy is a potential strategy to repair the damage through enhanced angiogenesis or by modulation of the inflammatory process via paracrine signaling. Amniotic fluid-derived progenitor cells (AFPCs) have been reported to differentiate into several lineages and can be used without ethical concerns or risk of teratoma formation. Since pigs are anatomically, physiologically, and genetically similar to humans, and pregnant pigs can be an abundant source of AFPCs, we used porcine AFPCs (pAFPCs) as our target cells. Intramyocardial injection of AFPCs has been shown to cure MI in animal models. However, intramuscular transplantation of cells has not been extensively investigated. In this study, we investigated the therapeutic potential of intramuscular injection of pAFPCs on acute MI. MI mice were divided into 1) PBS control, 2) medium cell dose (1 × 10(6) cells per leg; cell-M), and 3) high cell dose (4 × 10(6) cells per leg; cell-H) groups. Cells or PBS were directly injected into the hamstring muscle 20 min after MI surgery. Four weeks after MI surgery, the cell-M and cell-H groups exhibited significantly better ejection fraction, significantly greater wall thickness, smaller infarct scar sizes, and lower LV expansion index compared to the PBS group. Using in vivo imaging, we showed that the hamstring muscles from animals in the cell-M and cell-H groups had RFP-positive signals. In summary, intramuscular injection of porcine AFPCs reduced scar size, reduced pathological remodeling, and preserved heart function after MI. PMID:24667157

  10. STM and AFM; Which is Better for Surface Structural Analysis? Non- contact AFM Studies on Ge/Si(105) Surface

    NASA Astrophysics Data System (ADS)

    Hasegawa, Yukio

    2006-03-01

    Scanning tunneling microscopy (STM) has been utilized to determine surface atomic structure with its highly resolved images. Probing surface electronic states near the Fermi energy (EF), STM images, however, do not necessarily represent the atomic structure of surfaces. It has been believed that atomic force microscopy (AFM) provides us surface topographic images without being disturbed by the electronic states. In order to prove the surpassing performance, we performed noncontact (nc) AFM on the Ge/Si(105) surface [1], which is a facet plane of the ?hut? clusters formed on Ge-deposited Si(001) surface. It is found that STM images taken on the surface, either filled- or empty-state images, do not show all surface atoms because of the electronic effect; some surface atoms have dangling bond states below EF and other surface atoms have states above EF. [2]. In a nc-AFM image, on the other hand, all surface atoms having a dangling bond are observed [3], directly representing an atomic structure of the surface. Electronic information can also be obtained in AFM by using a Kelvin-probe method. From atomically resolved potential profile we obtained, charge transfer among the dangling bond states is directly demonstrated. These results clearly demonstrate that highly-resolved nc-AFM with a Kelvin-probe method is an ideal tool for analysis of atomic structures and electronic properties of surfaces. This work was done in collaboration with T. Eguchi, K. Akiyama, T. An, and M. Ono, ISSP, Univ. Tokyo and JST, Y. Fujikawa and T. Sakurai, IMR. Tohoku Univ. T. Hashimoto, AIST, Y. Morikawa, ISIR, Osaka Univ. K. Terakura, Hokkaido Univ., and M.G. Lagally, University of Wisconsin-Madison. [1] T. Eguchi et al., Phys. Rev. Lett. 93, 266102 (2004). [2] Y. Fujikawa et al., Phys. Rev. Lett. 88, 176101 (2002). [3] T. Eguchi and Y. Hasegawa, Phys. Rev. Lett. 89, 256105 (2002)

  11. Ciliary fluid transport enhanced by viscoelastic fluid

    NASA Astrophysics Data System (ADS)

    Guo, Hanliang; Kanso, Eva

    2015-11-01

    Motile cilia encounter complex, non-Newtonian fluids as they beat to gain self-propulsion of cells, transport fluids, and mix particles. Recently there have been many studies on swimming in complex fluids, both experimentally and theoretically. However the role of the non-Newtonian fluid in the ciliary transport system remains largely unknown. Here we use a one-way-coupled immersed boundary method to evaluate the impacts of viscoelastic fluid (Oldroyd-B fluid) on the fluid transport generated by an array of rabbit tracheal cilia beating in a channel at low Reynolds number. Our results show that the viscoelasticity could enhance the fluid transport generated by the rabbit tracheal cilia beating pattern and the flow is sensitive to the Deborah number in the range we investigate.

  12. Processing Technology in Microscopic Images of Cancer Cells in Pleural Fluid Based on Fuzzy Edge Detection Method

    NASA Astrophysics Data System (ADS)

    Zhang, L.; Wang, Q. G.; Qi, J. P.

    2006-10-01

    The traditional manual method of cancer cell recognizing requests a long period of diagnoses. In order to improve the efficiency and veracity of diagnoses, this article applies technology of image processing to analysis and recognition of cancer cell image. This article uses the fuzzy edge extraction, which is based on the OTSU threshold to process the original image of the cancer cells in pleural fluid, and then extracts the feature of the cancer cells according to the morphology automatically. The experiment shows that features such as area rate of karyon and cytoplasm, karyon division, shape of karyon and so on will provide evidences for diagnoses and will improve the efficiency and veracity of diagnoses.

  13. Multiparametric AFM reveals turgor-responsive net-like peptidoglycan architecture in live streptococci

    NASA Astrophysics Data System (ADS)

    Saar Dover, Ron; Bitler, Arkady; Shimoni, Eyal; Trieu-Cuot, Patrick; Shai, Yechiel

    2015-05-01

    Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.

  14. Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach

    PubMed Central

    Moschidou, Dafni; Mukherjee, Sayandip; Blundell, Michael P; Drews, Katharina; Jones, Gemma N; Abdulrazzak, Hassan; Nowakowska, Beata; Phoolchund, Anju; Lay, Kenneth; Ramasamy, T Selvee; Cananzi, Mara; Nettersheim, Daniel; Sullivan, Mark; Frost, Jennifer; Moore, Gudrun; Vermeesch, Joris R; Fisk, Nicholas M; Thrasher, Adrian J; Atala, Anthony; Adjaye, James; Schorle, Hubert; De Coppi, Paolo; Guillot, Pascale V

    2012-01-01

    Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling. PMID:22760542

  15. AFM-based mechanical characterization of single nanofibres.

    PubMed

    Neugirg, Benedikt R; Koebley, Sean R; Schniepp, Hannes C; Fery, Andreas

    2016-04-28

    Nanofibres are found in a broad variety of hierarchical biological systems as fundamental structural units, and nanofibrillar components are playing an increasing role in the development of advanced functional materials. Accurate determination of the mechanical properties of single nanofibres is thus of great interest, yet measurement of these properties is challenging due to the intricate specimen handling and the exceptional force and deformation resolution that is required. The atomic force microscope (AFM) has emerged as an effective, reliable tool in the investigation of nanofibrillar mechanics, with the three most popular approaches-AFM-based tensile testing, three-point deformation testing, and nanoindentation-proving preferable to conventional tensile testing in many (but not all) cases. Here, we review the capabilities and limitations of each of these methods and give a comprehensive overview of the recent advances in this field. PMID:27055900

  16. Insights into Epoxy Network Nanostructural Heterogeneity Using AFM-IR.

    PubMed

    Morsch, Suzanne; Liu, Yanwen; Lyon, Stuart B; Gibbon, Simon R

    2016-01-13

    The first direct observation of a chemically heterogeneous nanostructure within an epoxy resin is reported. Epoxy resins comprise the matrix component of many high performance composites, coatings and adhesives, yet the molecular network structure that underpins the performance of these industrially essential materials is not well understood. Internal nodular morphologies have repeatedly been reported for epoxy resins analyzed using SEM or AFM, yet the origin of these features remains a contentious subject, and epoxies are still commonly assumed to be chemically homogeneous. Uniquely, in this contribution we use the recently developed AFM-IR technique to eliminate previous differences in interpretation, and establish that nodule features correspond to heterogeneous network connectivity within an epoxy phenolic formulation. PMID:26694687

  17. AFM-based mechanical characterization of single nanofibres

    NASA Astrophysics Data System (ADS)

    Neugirg, Benedikt R.; Koebley, Sean R.; Schniepp, Hannes C.; Fery, Andreas

    2016-04-01

    Nanofibres are found in a broad variety of hierarchical biological systems as fundamental structural units, and nanofibrillar components are playing an increasing role in the development of advanced functional materials. Accurate determination of the mechanical properties of single nanofibres is thus of great interest, yet measurement of these properties is challenging due to the intricate specimen handling and the exceptional force and deformation resolution that is required. The atomic force microscope (AFM) has emerged as an effective, reliable tool in the investigation of nanofibrillar mechanics, with the three most popular approaches--AFM-based tensile testing, three-point deformation testing, and nanoindentation--proving preferable to conventional tensile testing in many (but not all) cases. Here, we review the capabilities and limitations of each of these methods and give a comprehensive overview of the recent advances in this field.

  18. Molecular modeling of enzyme attachment on AFM probes.

    PubMed

    Oliveira, Guedmiller S; Leite, Fabio L; Amarante, Adriano M; Franca, Eduardo F; Cunha, Richard A; Briggs, James M; Freitas, Luiz C G

    2013-09-01

    The immobilization of enzymes on atomic force microscope tip (AFM tip) surface is a crucial step in the development of nanobiosensors to be used in detection process. In this work, an atomistic modeling of the attachment of the acetyl coenzyme A carboxylase (ACC enzyme) on a functionalized AFM tip surface is proposed. Using electrostatic considerations, suitable enzyme-surface orientations with the active sites of the ACC enzyme available for interactions with bulk molecules were found. A 50 ns molecular dynamics trajectory in aqueous solution was obtained and surface contact area, hydrogen bonding and protein stability were analyzed. The enzyme-surface model proposed here with minor adjustment can be applied to study antigen-antibody interactions as well as enzyme immobilization on silica for chromatography applications. PMID:24029365

  19. AFM study of polymer lubricants on hard disk surfaces

    NASA Astrophysics Data System (ADS)

    Bao, G. W.; Troemel, M.; Li, S. F. Y.

    Thin liquid films of PFPE (perfluoropolyether) lubricants dip-coated on hard disk surfaces were imaged with non-contact mode AFM. Demnum lubricants with phosphazene additives exhibited strong interactions with a silicon tip due to the formation of liquid bridges between the lubricants and the tip, as indicated by a remarkable hysteresis loop between approach and retraction curves in force vs. distance measurements. Features resulting from capillary forces due to tip tapping to the lubricants were revealed, which demonstrated that the capillary forces could be used to lock the non-contacting tip at a certain separation from the substrate surface to obtain AFM images. Force vs. distance curves for Fomblin Z-dol lubricants showed negligible hysteresis effects and features corresponding to lateral distortion of the tip by the lubricants only were observed. In both cases, only when the tip was positioned far above the surfaces could the natural distributions of the lubricants be imaged.

  20. BOREAS AFM-5 Level-1 Upper Air Network Data

    NASA Technical Reports Server (NTRS)

    Barr, Alan; Hrynkiw, Charmaine; Newcomer, Jeffrey A. (Editor); Hall, Forrest G. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-5 team collected and processed data from the numerous radiosonde flights during the project. The goals of the AFM-05 team were to provide large-scale definition of the atmosphere by supplementing the existing Atmospheric Environment Service (AES) aerological network, both temporally and spatially. This data set includes basic upper-air parameters collected from the network of upper-air stations during the 1993, 1994, and 1996 field campaigns over the entire study region. The data are contained in tabular ASCII files. The level-1 upper-air network data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files also are available on a CD-ROM (see document number 20010000884).

  1. Adhesion forces in AFM of redox responsive polymer grafts: Effects of tip hydrophilicity

    NASA Astrophysics Data System (ADS)

    Feng, Xueling; Kieviet, Bernard D.; Song, Jing; Schön, Peter M.; Vancso, G. Julius

    2014-02-01

    The adherence between silicon nitride AFM tips and redox-active poly(ferrocenylsilanes) (PFS) grafts on gold was investigated by electrochemical AFM force spectroscopy. Before the adhesion measurements silicon nitride AFM probes were cleaned with organic solvents (acetone and ethanol) or piranha solution. Interestingly, these different AFM tip cleaning procedures drastically affected the observed adhesion forces. Water contact angle measurements on the corresponding AFM probe chips showed that piranha treatment resulted in a significant increase of AFM probe chip surface hydrophilicity compared to the organic solvent treatment. Obviously this hydrophilicity change caused drastic, even opposite changes in the tip-PFS adhesive force measurement upon electrode potential change to reversibly oxidize and reduce the PFS grafts. Our findings are of pivotal importance for AFM tip adhesion measurements utilizing standard silicon nitride AFM tips. Probe hydrophilicity must be carefully taken into consideration and controlled.

  2. A three-dimensional numerical simulation of cell behavior in a flow chamber based on fluid-solid interaction.

    PubMed

    Bai, Long; Cui, Yuhong; Zhang, Yixia; Zhao, Na

    2014-01-01

    The mechanical behavior of blood cells in the vessels has a close relationship with the physical characteristics of the blood and the cells. In this paper, a numerical simulation method was proposed to understand a single-blood cell's behavior in the vessels based on fluid-solid interaction method, which was conducted under adaptive time step and fixed time step, respectively. The main programme was C++ codes, which called FLUENT and ANSYS software, and UDF and APDL acted as a messenger to connect FLUENT and ANSYS for exchanging data. The computing results show: (1) the blood cell moved towards the bottom of the flow chamber in the beginning due to the influence of gravity, then it began to jump up when reached a certain height rather than touching the bottom. It could move downwards again after jump up, the blood cell could keep this way of moving like dancing continuously in the vessels; (2) the blood cell was rolling and deforming all the time; the rotation had oscillatory changes and the deformation became conspicuously when the blood cell was dancing. This new simulation method and results can be widely used in the researches of cytology, blood, cells, etc. PMID:25226968

  3. Human amniotic fluid stem cells labeled with up-conversion nanoparticles for imaging-monitored repairing of acute lung injury.

    PubMed

    Xu, Yunyun; Xiang, Jian; Zhao, He; Liang, Hansi; Huang, Jie; Li, Yan; Pan, Jian; Zhou, Huiting; Zhang, Xueguang; Wang, Jiang Huai; Liu, Zhuang; Wang, Jian

    2016-09-01

    Human amniotic fluid stem (hAFS) cells have generated a great deal of excitement in cell-based therapies and regenerative medicine. Here, we examined the effect of hAFS cells labeled with dual-polymer-coated UCNP-PEG-PEI nanoparticles in a murine model of acute lung injury (ALI). We observed hAFS cells migration to the lung using highly sensitive in vivo upconversion luminescence (UCL) imaging. We demonstrated that hAFS cells remained viable and retained their ability to differentiate even after UCNP-PEG-PEI labeling. More importantly, hAFS cells displayed remarkable positive effects on ALI-damaged lung tissue repair compared with mouse bone marrow mesenchymal stem cells (mBMSCs), which include recovery of the integrity of alveolar-capillary membrane, attenuation of transepithelial leukocyte and neutrophil migration, and down-regulation of proinflammatory cytokine and chemokine expression. Our work highlights a promising role for imaging-guided hAFS cell-based therapy in ALI. PMID:27244692

  4. AFM and SThM Characterization of Graphene

    NASA Astrophysics Data System (ADS)

    Foy, Christopher; Sidorov, Anton; Chen, Xunchi; Ruan, Ming; Berger, Claire; de Heer, Walter; Jiang, Zhigang

    2012-03-01

    We report on detailed characterization of epitaxial grown graphene on SiC and chemical vapor deposition grown graphene on Cu foil using atomic force microscopy (AFM) and scanning thermal microscopy (SThM). We focus on the electronic and thermal properties of graphene grain boundaries, and thus providing valuable feedback to materials growth. Specifically, we perform thermal conductivity contrast mapping and surface potential mapping of graphene, and compare with that obtained on the Au electrodes and the substrate.

  5. Investigation of biopolymer networks by means of AFM

    NASA Astrophysics Data System (ADS)

    Keresztes, Z.; Rigó, T.; Telegdi, J.; Kálmán, E.

    Natural hydrogel alginate was investigated by means of atomic force microscopy (AFM) to gain microscale information on the morphological and rheological properties of the biopolymer network cross-linked by various cations. Local rheological properties of the gels measured by force spectroscopy gave correlation between increasing ion selectivity and increasing polymer elasticity. Adhesive forces acting between the surface of the gel and the probe, and also the intrinsic rheological properties of bulk polymers affect the microscopical image formation.

  6. Near-Field Spectroscopy with Nanoparticles Deposited by AFM

    NASA Technical Reports Server (NTRS)

    Anderson, Mark S.

    2008-01-01

    An alternative approach to apertureless near-field optical spectroscopy involving an atomic-force microscope (AFM) entails less complexity of equipment than does a prior approach. The alternative approach has been demonstrated to be applicable to apertureless near-field optical spectroscopy of the type using an AFM and surface enhanced Raman scattering (SERS), and is expected to be equally applicable in cases in which infrared or fluorescence spectroscopy is used. Apertureless near-field optical spectroscopy is a means of performing spatially resolved analyses of chemical compositions of surface regions of nanostructured materials. In apertureless near-field spectroscopy, it is common practice to utilize nanostructured probe tips or nanoparticles (usually of gold) having shapes and dimensions chosen to exploit plasmon resonances so as to increase spectroscopic-signal strengths. To implement the particular prior approach to which the present approach is an alternative, it is necessary to integrate a Raman spectrometer with an AFM and to utilize a special SERS-active probe tip. The resulting instrumentation system is complex, and the tasks of designing and constructing the system and using the system to acquire spectro-chemical information from nanometer-scale regions on a surface are correspondingly demanding.

  7. Tissue section AFM: In situ ultrastructural imaging of native biomolecules

    PubMed Central

    Graham, Helen K.; Hodson, Nigel W.; Hoyland, Judith A.; Millward-Sadler, Sarah J.; Garrod, David; Scothern, Anthea; Griffiths, Christopher E.M.; Watson, Rachel E.B.; Cox, Thomas R.; Erler, Janine T.; Trafford, Andrew W.; Sherratt, Michael J.

    2010-01-01

    Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae. PMID:20144712

  8. Nanoscale rippling on polymer surfaces induced by AFM manipulation

    PubMed Central

    2015-01-01

    Summary Nanoscale rippling induced by an atomic force microscope (AFM) tip can be observed after performing one or many scans over the same area on a range of materials, namely ionic salts, metals, and semiconductors. However, it is for the case of polymer films that this phenomenon has been widely explored and studied. Due to the possibility of varying and controlling various parameters, this phenomenon has recently gained a great interest for some technological applications. The advent of AFM cantilevers with integrated heaters has promoted further advances in the field. An alternative method to heating up the tip is based on solvent-assisted viscoplastic deformations, where the ripples develop upon the application of a relatively low force to a solvent-rich film. An ensemble of AFM-based procedures can thus produce nanoripples on polymeric surfaces quickly, efficiently, and with an unprecedented order and control. However, even if nanorippling has been observed in various distinct modes and many theoretical models have been since proposed, a full understanding of this phenomenon is still far from being achieved. This review aims at summarizing the current state of the art in the perspective of achieving control over the rippling process on polymers at a nanoscale level. PMID:26733086

  9. Oxide nanocrystal based nanocomposites for fabricating photoplastic AFM probes.

    PubMed

    Ingrosso, Chiara; Martin-Olmos, Cristina; Llobera, Andreu; Innocenti, Claudia; Sangregorio, Claudio; Striccoli, Marinella; Agostiano, Angela; Voigt, Anja; Gruetzner, Gabi; Brugger, Jürgen; Perez-Murano, Francesc; Curri, Maria Lucia

    2011-11-01

    We report on the synthesis, characterization and application of a novel nanocomposite made of a negative tone epoxy based photoresist modified with organic-capped Fe(2)O(3) nanocrystals (NCs). The mechanical properties of the nanocomposite drastically improve upon incorporation of a suitable concentration of NCs in the polymer, without deteriorating its photolithography performance. High aspect ratio 3D microstructures made of the nanocomposite have been fabricated with a uniform surface morphology and with a resolution down to few micrometres. The embedded organic-capped Fe(2)O(3) NCs drastically increase the stiffness and hardness of the epoxy based photoresist matrix, making the final material extremely interesting for manufacturing miniaturized polymer based mechanical devices and systems. In particular, the nanocomposite has been used as structural material for fabricating photoplastic Atomic Force Microscopy (AFM) probes with integrated tips showing outstanding mechanical response and high resolution imaging performance. The fabricated probes consist of straight cantilevers with low stress-gradient and high quality factors, incorporating sharp polymeric tips. They present considerably improved performance compared to pure epoxy based photoresist AFM probes, and to commercial silicon AFM probes. PMID:21858377

  10. In vitro fabrication of autologous living tissue-engineered vascular grafts based on prenatally harvested ovine amniotic fluid-derived stem cells.

    PubMed

    Weber, Benedikt; Kehl, Debora; Bleul, Ulrich; Behr, Luc; Sammut, Sébastien; Frese, Laura; Ksiazek, Agnieszka; Achermann, Josef; Stranzinger, Gerald; Robert, Jérôme; Sanders, Bart; Sidler, Michele; Brokopp, Chad E; Proulx, Steven T; Frauenfelder, Thomas; Schoenauer, Roman; Emmert, Maximilian Y; Falk, Volkmar; Hoerstrup, Simon P

    2016-01-01

    Amniotic fluid cells (AFCs) have been proposed as a valuable source for tissue engineering and regenerative medicine. However, before clinical implementation, rigorous evaluation of this cell source in clinically relevant animal models accepted by regulatory authorities is indispensable. Today, the ovine model represents one of the most accepted preclinical animal models, in particular for cardiovascular applications. Here, we investigate the isolation and use of autologous ovine AFCs as cell source for cardiovascular tissue engineering applications. Fetal fluids were aspirated in vivo from pregnant ewes (n = 9) and from explanted uteri post mortem at different gestational ages (n = 91). Amniotic non-allantoic fluid nature was evaluated biochemically and in vivo samples were compared with post mortem reference samples. Isolated cells revealed an immunohistochemical phenotype similar to ovine bone marrow-derived mesenchymal stem cells (MSCs) and showed expression of stem cell factors described for embryonic stem cells, such as NANOG and STAT-3. Isolated ovine amniotic fluid-derived MSCs were screened for numeric chromosomal aberrations and successfully differentiated into several mesodermal phenotypes. Myofibroblastic ovine AFC lineages were then successfully used for the in vitro fabrication of small- and large-diameter tissue-engineered vascular grafts (n = 10) and cardiovascular patches (n = 34), laying the foundation for the use of this relevant pre-clinical in vivo assessment model for future amniotic fluid cell-based therapeutic applications. PMID:23881794

  11. Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage

    PubMed Central

    Qin, Mingde; Chen, Ruihua; Li, Hong; Liang, Hansi; Xue, Qun; Li, Fang; Chen, Ying; Zhang, Xueguang

    2016-01-01

    Amniotic fluid stem cells (AFSCs) are a type of fetal stem cell whose stemness encompasses both embryonic and adult stem cells, suggesting that they may be easily and efficiently reprogrammed into induced pluripotent stem cells (iPSCs). To further simplify the reprogramming process, the creation of AFSC-derived iPSCs using a single factor is desirable. Here we report the generation of one-factor human AFSC-iPSCs (AiPSCs) from human AFSCs by ectopic expression of the transcription factor OCT4. Just like human embryonic stem cells, AiPSCs exhibited similar epigenetic status, global gene expression profiles, teratoma formation and in vitro & in vivo pluripotency. Our results indicate that the OCT4 is necessary and sufficient to directly reprogram human AFSCs into pluripotent AiPSCs. Moreover, reflecting the similar memory characteristics of AFSCs and neural stem cells, we show that AiPSC membrane-derived vesicles (MVs) repair cerebral ischemia damage. We anticipate that the successful generation of one-factor AiPSCs will facilitate the creation of patient-specific pluripotent stem cells without the need for transgenic expression of oncogenes. Moreover, MVs from tissue-specific AiPSCs have potential in tissue repair, representing a novel application of iPSCs. PMID:27019637

  12. Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors

    PubMed Central

    Le Cleach, L; Delaire, S; Boumsell, L; Bagot, M; Bourgault-Villada, I; Bensussan, A; Roujeau, J C

    2000-01-01

    Toxic epidermal necrolysis (TEN) is a rare life-threatening adverse drug reaction characterized by a massive destruction of the epidermis. Immunohistological studies of skin biopsies of TEN showed infiltrates of predominantly CD8+ T lymphocytes even though other authors reported a prominent involvement of cells of the monocyte-macrophage lineage. The aim of this study was to characterize phenotypically and functionally the cells present in the cutaneous blister fluid of four patients with TEN. We first determined that lymphocytes were predominant in blister fluid obtained early, while monocytes/macrophages later became the most important population. We then showed that this lymphocyte population, mainly CD3+CD8+, corresponded to a peculiar cell subset as they expressed cutaneous leucocyte antigen, killer inhibitory receptors KIR/KAR and failed to express CD28 molecule. Functionally, we determined that blister T lymphocytes had a cytotoxic T lymphocyte (CTL)- and NK-like cytotoxicity. The role of this cytotoxic lymphocyte population present at the site of lesions during TEN remains to be understood. PMID:10606987

  13. A new parallel plate shear cell for in situ real-space measurements of complex fluids under shear flow

    NASA Astrophysics Data System (ADS)

    Wu, Yu Ling; Brand, Joost H. J.; van Gemert, Josephus L. A.; Verkerk, Jaap; Wisman, Hans; van Blaaderen, Alfons; Imhof, Arnout

    2007-10-01

    We developed and tested a parallel plate shear cell that can be mounted on top of an inverted microscope to perform confocal real-space measurements on complex fluids under shear. To follow structural changes in time, a plane of zero velocity is created by letting the plates move in opposite directions. The location of this plane is varied by changing the relative velocities of the plates. The gap width is variable between 20 and 200μm with parallelism better than 1μm. Such a small gap width enables us to examine the total sample thickness using high numerical aperture objective lenses. The achieved shear rates cover the range of 0.02-103s-1. This shear cell can apply an oscillatory shear with adjustable amplitude and frequency. The maximum travel of each plate equals 1cm, so that strains up to 500 can be applied. For most complex fluids, an oscillatory shear with such a large amplitude can be regarded as a continuous shear. We measured the flow profile of a suspension of silica colloids in this shear cell. It was linear except for a small deviation caused by sedimentation. To demonstrate the excellent performance and capabilities of this new setup we examined shear induced crystallization and melting of concentrated suspensions of 1μm diameter silica colloids.

  14. A new parallel plate shear cell for in situ real-space measurements of complex fluids under shear flow.

    PubMed

    Wu, Yu Ling; Brand, Joost H J; van Gemert, Josephus L A; Verkerk, Jaap; Wisman, Hans; van Blaaderen, Alfons; Imhof, Arnout

    2007-10-01

    We developed and tested a parallel plate shear cell that can be mounted on top of an inverted microscope to perform confocal real-space measurements on complex fluids under shear. To follow structural changes in time, a plane of zero velocity is created by letting the plates move in opposite directions. The location of this plane is varied by changing the relative velocities of the plates. The gap width is variable between 20 and 200 microm with parallelism better than 1 microm. Such a small gap width enables us to examine the total sample thickness using high numerical aperture objective lenses. The achieved shear rates cover the range of 0.02-10(3) s(-1). This shear cell can apply an oscillatory shear with adjustable amplitude and frequency. The maximum travel of each plate equals 1 cm, so that strains up to 500 can be applied. For most complex fluids, an oscillatory shear with such a large amplitude can be regarded as a continuous shear. We measured the flow profile of a suspension of silica colloids in this shear cell. It was linear except for a small deviation caused by sedimentation. To demonstrate the excellent performance and capabilities of this new setup we examined shear induced crystallization and melting of concentrated suspensions of 1 microm diameter silica colloids. PMID:17979430

  15. The Hydrothermal Diamond Anvil Cell (HDAC) for raman spectroscopic studies of geologic fluids at high pressures and temperatures

    USGS Publications Warehouse

    Schmidt, Christian; Chou, I-Ming

    2012-01-01

    In this chapter, we describe the hydrothermal diamond-anvil cell (HDAC), which is specifically designed for experiments on systems with aqueous fluids to temperatures up to ⬚~1000ºC and pressures up to a few GPa to tens of GPa. This cell permits optical observation of the sample and the in situ determination of properties by ‘photon-in photon-out’ techniques such as Raman spectroscopy. Several methods for pressure measurement are discussed in detail including the Raman spectroscopic pressure sensors a-quartz, berlinite, zircon, cubic boron nitride (c-BN), and 13C-diamond, the fluorescence sensors ruby (α-Al2O3:Cr3+), Sm:YAG (Y3Al5O12:Sm3+) and SrB4O7:Sm2+, and measurements of phase-transition temperatures. Furthermore, we give an overview of published Raman spectroscopic studies of geological fluids to high pressures and temperatures, in which diamond anvil cells were applied.

  16. Chapter 7: The hydrothermal diamond anvil cell (HDAC) for Raman spectroscopic studies of geological fluids at high pressures and temperatures

    USGS Publications Warehouse

    Schmidt, Christian; Chou, I-Ming

    2012-01-01

    In this chapter, we describe the hydrothermal diamond-anvil cell (HDAC), which is specifically designed for experiments on systems with aqueous fluids to temperatures up to ~1000ºC and pressures up to a few GPa to tens of GPa. This cell permits optical observation of the sample and the in situ determination of properties by ‘photon-in photon-out’ techniques such as Raman spectroscopy. Several methods for pressure measurement are discussed in detail including the Raman spectroscopic pressure sensors a-quartz, berlinite, zircon, cubic boron nitride (c-BN), and 13C-diamond, the fluorescence sensors ruby (α-Al2O3:Cr3+), Sm:YAG (Y3Al5O12:Sm3+) and SrB4O7:Sm2+, and measurements of phase-transition temperatures. Furthermore, we give an overview of published Raman spectroscopic studies of geological fluids to high pressures and temperatures, in which diamond anvil cells were applied.

  17. T cell acute lymphoblastic leukaemia presenting with sudden onset right oculomotor nerve palsy with normal neuroradiography and cerebrospinal fluid studies

    PubMed Central

    Bhatt, Vijaya Raj; Naqi, Muniba; Bartaula, Rajiv; Murukutla, Srujitha; Misra, Sulagna; Popalzai, Muhammad; Paramanathan, Kavitha; Dai, Qun

    2012-01-01

    Leptomeningeal disease presenting with neurological dysfunction is not uncommon in leukaemia. However, it is often accompanied by abnormalities in cerebrospinal fluid (CSF) studies and/or neuroradiography. Here, the authors describe a case of a young patient presenting with sudden onset right oculomotor nerve palsy with normal neuroradiography and CSF studies, who was subsequently diagnosed to have T cell acute lymphoblastic leukaemia (T-ALL). This case highlights that neurological manifestations can be the initial presenting feature of T-ALL and can occur suddenly despite normal neuroradiography and initial CSF studies. PMID:22605802

  18. Comparison of multi-fluid moment models with particle-in-cell simulations of collisionless magnetic reconnection

    SciTech Connect

    Wang, Liang Germaschewski, K.; Hakim, Ammar H.; Bhattacharjee, A.

    2015-01-15

    We introduce an extensible multi-fluid moment model in the context of collisionless magnetic reconnection. This model evolves full Maxwell equations and simultaneously moments of the Vlasov-Maxwell equation for each species in the plasma. Effects like electron inertia and pressure gradient are self-consistently embedded in the resulting multi-fluid moment equations, without the need to explicitly solving a generalized Ohm's law. Two limits of the multi-fluid moment model are discussed, namely, the five-moment limit that evolves a scalar pressures for each species and the ten-moment limit that evolves the full anisotropic, non-gyrotropic pressure tensor for each species. We first demonstrate analytically and numerically that the five-moment model reduces to the widely used Hall magnetohydrodynamics (Hall MHD) model under the assumptions of vanishing electron inertia, infinite speed of light, and quasi-neutrality. Then, we compare ten-moment and fully kinetic particle-in-cell (PIC) simulations of a large scale Harris sheet reconnection problem, where the ten-moment equations are closed with a local linear collisionless approximation for the heat flux. The ten-moment simulation gives reasonable agreement with the PIC results regarding the structures and magnitudes of the electron flows, the polarities and magnitudes of elements of the electron pressure tensor, and the decomposition of the generalized Ohm's law. Possible ways to improve the simple local closure towards a nonlocal fully three-dimensional closure are also discussed.

  19. Comparison of multi-fluid moment models with particle-in-cell simulations of collisionless magnetic reconnection

    NASA Astrophysics Data System (ADS)

    Wang, Liang; Hakim, Ammar H.; Bhattacharjee, A.; Germaschewski, K.

    2015-01-01

    We introduce an extensible multi-fluid moment model in the context of collisionless magnetic reconnection. This model evolves full Maxwell equations and simultaneously moments of the Vlasov-Maxwell equation for each species in the plasma. Effects like electron inertia and pressure gradient are self-consistently embedded in the resulting multi-fluid moment equations, without the need to explicitly solving a generalized Ohm's law. Two limits of the multi-fluid moment model are discussed, namely, the five-moment limit that evolves a scalar pressures for each species and the ten-moment limit that evolves the full anisotropic, non-gyrotropic pressure tensor for each species. We first demonstrate analytically and numerically that the five-moment model reduces to the widely used Hall magnetohydrodynamics (Hall MHD) model under the assumptions of vanishing electron inertia, infinite speed of light, and quasi-neutrality. Then, we compare ten-moment and fully kinetic particle-in-cell (PIC) simulations of a large scale Harris sheet reconnection problem, where the ten-moment equations are closed with a local linear collisionless approximation for the heat flux. The ten-moment simulation gives reasonable agreement with the PIC results regarding the structures and magnitudes of the electron flows, the polarities and magnitudes of elements of the electron pressure tensor, and the decomposition of the generalized Ohm's law. Possible ways to improve the simple local closure towards a nonlocal fully three-dimensional closure are also discussed.

  20. Vacuum-assisted Fluid Flow in Microchannels to Pattern Substrates and Cells

    PubMed Central

    Shrirao, Anil B.; Kung, Frank H.; Yip, Derek; Cho, Cheul H.; Townes-Anderson, Ellen

    2014-01-01

    Substrate and cell patterning are widely used techniques in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This paper describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. Our method builds upon a previous vacuum-assisted method used for micromolding (Jeon, Choi et al. 1999) and successfully patterned collagen-I, fibronectin and Sal-1 substrates on glass and polystyrene surfaces, filling microchannels with lengths up to 120 mm and covering areas up to 13 × 10 mm2. Vacuum-patterned substrates were subsequently used to culture mammalian PC12 and fibroblast cells and amphibian neurons. Cells were also patterned directly by injecting cell suspensions into microchannels using vacuum. Fibroblast and neuronal cells patterned using vacuum showed normal growth and minimal cell death indicating no adverse effects of vacuum on cells. Our method fills reversibly sealed PDMS microchannels. This enables the user to remove the PDMS microchannel cast and access the patterned biomaterial or cells for further experimental purposes. Overall, this is a straightforward technique that has broad applicability for cell biology. PMID:24989641

  1. Generation of Induced Pluripotent Stem Cells from Human Amniotic Fluid Cells by Reprogramming with Two Factors in Feeder-free Conditions

    PubMed Central

    LI, Qing; FAN, Yong; SUN, Xiaofang; YU, Yanhong

    2012-01-01

    Abstract The ectopic expression of transcription factors for reprogramming human somatic cells to a pluripotent state represents a valuable resource for the development of in vitro-based models for human disease and has great potential in regenerative therapies. However, the majority of studies have used skin fibroblasts to generate induced pluripotent stem cells (iPSCs) that typically require the enforced expression of several transcription factors, thereby posing a mutagenesis risk by the insertion of viral transgenes. To reduce this risk, iPSCs have been generated with OCT4 and KLF4 from human neural stem cells that endogenously express the remaining reprogramming factors. However, human neural stem cells are rare and difficult to obtain. Here, we show that iPSCs can be generated from human amniotic fluid cells (hAFCs) with two transcription factors: OCT4 and KLF4. Furthermore, iPSCs can be readily derived from hAFCs in a feeder-free conditions, thereby eliminating the potential variability caused by using feeder cells. Our results indicate that hAFCs represent an accessible source of cells that can be reprogrammed into pluripotent stem cells with two Yamanaka factors. Therefore, hAFCs may become a preferred cell type in the future for safe reprogramming without any exogenous genetic material. PMID:23138118

  2. mTORC1 Is Essential for Early Steps during Schwann Cell Differentiation of Amniotic Fluid Stem Cells and Regulates Lipogenic Gene Expression

    PubMed Central

    Schörghofer, David; Kinslechner, Katharina; Schütz, Birgit; Thi Thanh Pham, Ha; Rosner, Margit; Joo, Gabor Jozsef; Röhrl, Clemens; Weichhart, Thomas; Stangl, Herbert; Lubec, Gert; Hengstschläger, Markus; Mikula, Mario

    2014-01-01

    Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS) cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR) pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1) activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway. PMID:25221943

  3. Characterisation of synovial fluid and infrapatellar fat pad derived mesenchymal stromal cells: The influence of tissue source and inflammatory stimulus

    PubMed Central

    Garcia, John; Wright, Karina; Roberts, Sally; Kuiper, Jan Herman; Mangham, Chas; Richardson, James; Mennan, Claire

    2016-01-01

    The infrapatellar fat pad (FP) and synovial fluid (SF) in the knee serve as reservoirs of mesenchymal stromal cells (MSCs) with potential therapeutic benefit. We determined the influence of the donor on the phenotype of donor matched FP and SF derived MSCs and examined their immunogenic and immunomodulatory properties before and after stimulation with the pro-inflammatory cytokine interferon-gamma (IFN-γ). Both cell populations were positive for MSC markers CD73, CD90 and CD105, and displayed multipotency. FP-MSCs had a significantly faster proliferation rate than SF-MSCs. CD14 positivity was seen in both FP-MSCs and SF-MSCs, and was positively correlated to donor age but only for SF-MSCs. Neither cell population was positive for the co-stimulatory markers CD40, CD80 and CD86, but both demonstrated increased levels of human leukocyte antigen-DR (HLA-DR) following IFN-γ stimulation. HLA-DR production was positively correlated with donor age for FP-MSCs but not SF-MSCs. The immunomodulatory molecule, HLA-G, was constitutively produced by both cell populations, unlike indoleamine 2, 3-dioxygenase which was only produced following IFN-γ stimulation. FP and SF are accessible cell sources which could be utilised in the treatment of cartilage injuries, either by transplantation following ex-vivo expansion or endogenous targeting and mobilisation of cells close to the site of injury. PMID:27073003

  4. The potential of mesenchymal stem cells derived from amniotic membrane and amniotic fluid for neuronal regenerative therapy

    PubMed Central

    Kim, Eun Young; Lee, Kyung-Bon; Kim, Min Kyu

    2014-01-01

    The mesenchymal stem cells (MSCs), which are derived from the mesoderm, are considered as a readily available source for tissue engineering. They have multipotent differentiation capacity and can be differentiated into various cell types. Many studies have demonstrated that the MSCs identified from amniotic membrane (AM-MSCs) and amniotic fluid (AF-MSCs) are shows advantages for many reasons, including the possibility of noninvasive isolation, multipotency, self-renewal, low immunogenicity, anti-inflammatory and nontumorigenicity properties, and minimal ethical problem. The AF-MSCs and AM-MSCs may be appropriate sources of mesenchymal stem cells for regenerative medicine, as an alternative to embryonic stem cells (ESCs). Recently, regenerative treatments such as tissue engineering and cell transplantation have shown potential in clinical applications for degenerative diseases. Therefore, amnion and MSCs derived from amnion can be applied to cell therapy in neuro-degeneration diseases. In this review, we will describe the potential of AM-MSCs and AF-MSCs, with particular focus on cures for neuronal degenerative diseases. [BMB Reports 2014; 47(3): 135-140] PMID:24499672

  5. Monitoring the Evaporation of Fluids from Fiber-Optic Micro-Cell Cavities

    PubMed Central

    Preter, Eyal; Preloznik, Borut; Artel, Vlada; Sukenik, Chaim N.; Donlagic, Denis; Zadok, Avi

    2013-01-01

    Fiber-optic sensors provide remote access, are readily embedded within structures, and can operate in harsh environments. Nevertheless, fiber-optic sensing of liquids has been largely restricted to measurements of refractive index and absorption spectroscopy. The temporal dynamics of fluid evaporation have potential applications in monitoring the quality of water, identification of fuel dilutions, mobile point-of-care diagnostics, climatography and more. In this work, the fiber-optic monitoring of fluids evaporation is proposed and demonstrated. Sub-nano-liter volumes of a liquid are applied to inline fiber-optic micro-cavities. As the liquid evaporates, light is refracted out of the cavity at the receding index boundary between the fluid and the ambient surroundings. A sharp transient attenuation in the transmission of light through the cavity, by as much as 50 dB and on a sub-second time scale, is observed. Numerical models for the transmission dynamics in terms of ray-tracing and wavefront propagation are provided. Experiments show that the temporal transmission profile can distinguish between different liquids. PMID:24212122

  6. Immune Activation and HIV-Specific CD8(+) T Cells in Cerebrospinal Fluid of HIV Controllers and Noncontrollers.

    PubMed

    Ganesh, Anupama; Lemongello, Donna; Lee, Evelyn; Peterson, Julia; McLaughlin, Bridget E; Ferre, April L; Gillespie, Geraldine M; Fuchs, Dietmar; Deeks, Steven G; Hunt, Peter W; Price, Richard W; Spudich, Serena S; Shacklett, Barbara L

    2016-08-01

    The central nervous system (CNS) is an important target of HIV, and cerebrospinal fluid (CSF) can provide a window into host-virus interactions within the CNS. The goal of this study was to determine whether HIV-specific CD8(+) T cells are present in CSF of HIV controllers (HC), who maintain low to undetectable plasma viremia without antiretroviral therapy (ART). CSF and blood were sampled from 11 HC, defined based on plasma viral load (VL) consistently below 2,000 copies/ml without ART. These included nine elite controllers (EC, plasma VL <40 copies/ml) and two viremic controllers (VC, VL 40-2,000 copies/ml). All controllers had CSF VL <40 copies/ml. Three comparison groups were also sampled: six HIV noncontrollers (NC, VL >10,000 copies/ml, no ART); seven individuals with viremia suppressed due to ART (Tx, VL <40 copies/ml); and nine HIV-negative controls. CD4(+) and CD8(+) T cells in CSF and blood were analyzed by flow cytometry to assess expression of CCR5, activation markers CD38 and HLA-DR, and memory/effector markers CD45RA and CCR7. HIV-specific CD8(+) T cells were quantified by major histocompatibility complex class I multimer staining. HIV-specific CD8(+) T cells were detected ex vivo at similar frequencies in CSF of HC and noncontrollers; the highest frequencies were in individuals with CD4 counts below 500 cells/μl. The majority of HIV-specific CD8(+) T cells in CSF were effector memory cells expressing CCR5. Detection of these cells in CSF suggests active surveillance of the CNS compartment by HIV-specific T cells, including in individuals with long-term control of HIV infection in the absence of therapy. PMID:27019338

  7. Analytical and Numerical Modeling of Fluid Flow and Heat Transfer through Open-Cell Metal Foam Heat Exchangers

    NASA Astrophysics Data System (ADS)

    Taheri, Mehrdad

    In this thesis analytical and numerical investigations of fluid flow and heat transfer through open cell metal foam heat exchangers are presented. Primarily, different representative unit cell approximations, i.e, tetrakaidecahedron, dodecahedron and cubic are discussed. By applying the thermal resistance analogy, a novel formulation for evaluation of the effective thermal conductivity of metal foams is proposed. The model improves previous models based on cubic or hexagonal cells. By using computer tomography images of a nickel foam sample a realistic 3D geometry is created and the foam's geometrical properties (i.e., porosity and surface area to volume ratio) and effective thermal conductivity are obtained. By using the experimentally found values of permeability, Forchheimer coefficient and solid-fluid interfacial convection coefficient, mathematical models for fluid flow and heat transfer in metal foams are developed. Two different assumptions: local thermal equilibrium (LTE) and local thermal non-equilibrium (LTNE), are used. LTNE yields more accurate results. A three-dimensional computational fluid dynamics (CFD) model of metal foam is made and validated against the experimental data for a square cross sectional nickel foam heat exchanger channel heated from the side walls while cooling air passes through the foam. The simulations are carried out for constant temperature or heat flux and different foam materials with pore densities of 10 and 40 pores per inch. The results show that the bonding of the foam to the walls has a considerable impact on the heat transfer rate. Convective heat transfer coefficients in terms of Nusselt number as functions of Reynolds number are also obtained. The design and CFD modeling of metal foam cross flow heat exchangers are also discussed. The results indicate both effectiveness and number of transfer units (NTU) for the metal foam heat exchangers are higher than those of a hollow channel; however, the effectiveness-NTU curves

  8. Comparative study of CD4 and CD45RO T cells and CD20 B cells in cerebrospinal fluid of syphilitic meningitis and tuberculous meningitis patients.

    PubMed

    Yu, Nian; Zhang, Qiao-Quan; Zhang, Kang; Xie, Yuan; Zhu, Hai-Qing; Lin, Xing-Jian; Di, Qing

    2016-09-01

    This study was to investigate the differences of lymphocyte in the cerebrospinal fluid (CSF) of patients with syphilis meningitis (SM) and tuberculous meningitis (TBM) for new diagnostic insights. Totally, 79 cases of SM and 45 cases of TBM were enrolled. In the CSF, the CD4, CD45RO or CD20 positive lymphocytes were detected by immunohistochemistry. The proportion of CD4 T cells in the CSF lymphocytes in patients with SM was significantly higher than that in patients with TBM (p < 0.05). After medical therapy, there was a significantly decline trend of the CD4 T-cell proportion in both groups (p < 0.05). The proportion of CD45RO T cells in CSF lymphocytes of patients with SM was less than that of patients with TBM (p < 0.05). After medical therapy, the positive ratio of CD45RO T cells was increased in the CSF of both group patients (p < 0.05). The proportion of CD20B cells in the CSF lymphocytes was not obviously different between the two groups during every stage. In conclusion, there are strong differences of CD4 and CD45RO T-cell ratio, but not the CD20 B cells in the meningitis. CD4 and CD45RO T cells in CSF are a useful complement in differentially diagnosing SM and TBM; it contributes to further understand the pathogenesis and prognosis of SM and TBM. PMID:27467195

  9. HCO3(-) secretion by murine nasal submucosal gland serous acinar cells during Ca2+-stimulated fluid secretion.

    PubMed

    Lee, Robert J; Harlow, Janice M; Limberis, Maria P; Wilson, James M; Foskett, J Kevin

    2008-07-01

    Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl(-) content revealed by cell shrinkage, reflecting Cl(-) secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na(+)/H(+) exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3

  10. SR-XRD in situ monitoring of copper-IUD corrosion in simulated uterine fluid using a portable spectroelectrochemical cell.

    PubMed

    Grayburn, Rosie A; Dowsett, Mark G; Sabbe, Pieter-Jan; Wermeille, Didier; Anjos, Jorge Alves; Flexer, Victoria; De Keersmaecker, Michel; Wildermeersch, Dirk; Adriaens, Annemie

    2016-08-01

    The objective of this work is to study the initial corrosion of copper in the presence of gold when placed in simulated uterine fluid in order to better understand the evolution of active components of copper-IUDs. In order to carry out this study, a portable cell was designed to partially simulate the uterine environment and provide a way of tracking the chemical changes occurring in the samples in situ within a controlled environment over a long period of time using synchrotron spectroelectrochemistry. The dynamically forming crystalline corrosion products are determined in situ for a range of copper-gold surface ratios over the course of a 10-day experiment in the cell. It is concluded that the insoluble deposits forming over this time are not the origin of the anticonception mechanism. PMID:27017519

  11. Solvent-mediated repair and patterning of surfaces by AFM

    SciTech Connect

    Elhadj, S; Chernov, A; De Yoreo, J

    2007-10-30

    A tip-based approach to shaping surfaces of soluble materials with nanometer-scale control is reported. The proposed method can be used, for example, to eliminate defects and inhomogeneities in surface shape, repair mechanical or laser-induced damage to surfaces, or perform 3D lithography on the length scale of an AFM tip. The phenomenon that enables smoothing and repair of surfaces is based on the transport of material from regions of high- to low-curvature within the solution meniscus formed in a solvent-containing atmosphere between the surface in question and an AFM tip scanned over the surface. Using in situ AFM measurements of the kinetics of surface remodeling on KDP (KH{sub 2}PO{sub 4}) crystals in humid air, we show that redistribution of solute material during relaxation of grooves and mounds is driven by a reduction in surface free energy as described by the Gibbs-Thomson law. We find that the perturbation from a flat interface evolves according to the diffusion equation where the effective diffusivity is determined by the product of the surface stiffness and the step kinetic coefficient. We also show that, surprisingly, if the tip is instead scanned over or kept stationary above an atomically flat area of the surface, a convex structure is formed with a diameter that is controlled by the dimensions of the meniscus, indicating that the presence of the tip and meniscus reduces the substrate chemical potential beneath that of the free surface. This allows one to create nanometer-scale 3D structures of arbitrary shape without the removal of substrate material or the use of extrinsic masks or chemical compounds. Potential applications of these tip-based phenomena are discussed.

  12. Applications of AFM for atomic manipulation and spectroscopy

    NASA Astrophysics Data System (ADS)

    Custance, Oscar

    2009-03-01

    Since the first demonstration of atom-by-atom assembly [1], atomic manipulation with scanning tunneling microscopy has yielded stunning realizations in nanoscience. A new exciting panorama has been recently opened with the possibility of manipulating atoms at surfaces using atomic force microscopy (AFM) [2-5]. In this talk, we will present two different approaches that enable patterning structures at semiconductor surfaces by manipulating individual atoms with AFM and at room temperature [2, 3]. We will discuss the physics behind each protocol through the analysis of the measured forces associated with these manipulations [3-5]. Another challenging issue in scanning probe microscopy is the ability to disclose the local chemical composition of a multi-element system at atomic level. Here, we will introduce a single-atom chemical identification method, which is based on detecting the forces between the outermost atom of the AFM tip and the atoms at a surface [6]. We demonstrate this identification procedure on a particularly challenging system, where any discrimination attempt based solely on topographic measurements would be impossible to achieve. [4pt] References: [0pt] [1] D. M. Eigler and E. K. Schweizer, Nature 344, 524 (1990); [0pt] [2] Y. Sugimoto, M. Abe, S. Hirayama, N. Oyabu, O. Custance and S. Morita, Nature Materials 4, 156 (2005); [0pt] [3] Y. Sugimoto, P. Pou, O. Custance, P. Jelinek, M. Abe, R. Perez and S. Morita, Science 322, 413 (2008); [0pt] [4] Y. Sugimoto, P. Jelinek, P. Pou, M. Abe, S. Morita, R. Perez and O. Custance, Phys. Rev. Lett. 98, 106104 (2007); [0pt] [5] M. Ternes, C. P. Lutz, C. F. Hirjibehedin, F. J. Giessibl and A. J. Heinrich, Science 319, 1066 (2008); [0pt] [6] Y. Sugimoto, P. Pou, M. Abe, P. Jelinek, R. Perez, S. Morita, and O. Custance, Nature 446, 64 (2007)

  13. A Comprehensive Fluid Dynamic-Diffusion Model of Blood Microcirculation with Focus on Sickle Cell Disease

    NASA Astrophysics Data System (ADS)

    Le Floch, Francois; Harris, Wesley L.

    2009-11-01

    A novel methodology has been developed to address sickle cell disease, based on highly descriptive mathematical models for blood flow in the capillaries. Our investigations focus on the coupling between oxygen delivery and red blood cell dynamics, which is crucial to understanding sickle cell crises and is unique to this blood disease. The main part of our work is an extensive study of blood dynamics through simulations of red cells deforming within the capillary vessels, and relies on the use of a large mathematical system of equations describing oxygen transfer, blood plasma dynamics and red cell membrane mechanics. This model is expected to lead to the development of new research strategies for sickle cell disease. Our simulation model could be used not only to assess current researched remedies, but also to spur innovative research initiatives, based on our study of the physical properties coupled in sickle cell disease.

  14. A cryogenic high pressure cell for inelastic neutron scattering measurements of quantum fluids and solids.

    PubMed

    Carmichael, J R; Diallo, S O

    2013-01-01

    We present our new development of a high pressure cell for inelastic neutron scattering measurements of helium at ultra-low temperatures. The cell has a large sample volume of ~140 cm(3) and a working pressure of ~7 MPa, with a relatively thin wall-thickness (1.1 mm)--thanks to the high yield strength aluminum used in the design. Two variants of this cell have been developed. The first cell is permanently joined components using electron-beam welding and explosion welding, methods that have little or no impact on the global heat treatment of the cell. The second cell discussed has modular and interchangeable components, which includes a capacitance pressure gauge, that can be sealed using the traditional indium wire technique. The performance of the cells have been tested in recent measurements on superfluid liquid helium near the solidification line. PMID:23387689

  15. Influence of different fluid resuscitation techniques on the number of myeloid-derived suppressor cells in rats.

    PubMed

    Wang, Z J; Wang, H X; Li, L; Wang, L; Dou, H H

    2016-01-01

    We investigated the influence of different fluid resuscitation techniques on the number of myeloid-derived suppressor cells (MDSCs) in rats. Seventy-two healthy Sprague-Dawley rats were randomly divided into groups that received sham operation (Sham group), hypertonic saline (HRS group), lactated ringer's solution (LRS group), or crystalloid solution (LCRS group). Six rats from each group were sacrificed by cervical dislocation at 12, 24, and 48 h after resuscitation. The spleens were harvested under sterile conditions and spleen cell suspension was prepared. The number of MDSCs was detected using flow cytometry. The number of MDSCs in the Sham group did not differ significantly among the different time points. Compared with the Sham group, the number of MDSCs after the use of the different fluid resuscitation techniques increased to varying extents and the differences among the groups were significant. The number of MDSCs in the HRS group was much lower than that of the LRS and LCRS groups at both 24 and 48 h (P < 0.05). At 12 h, the number of MDSCs in the HRS group was significantly lower than that of the LRS group (P < 0.05). The differences between the HRS and LCRS groups were not statistically significant. Shortly after hemorrhagic shock resuscitation, the immune function of rats was suppressed to a varying extent and was gradually restored over time. Resuscitation with HRS alleviated the immunosuppression at the early stage after shock. PMID:27173299

  16. Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance in human lungs rejected for transplantation

    PubMed Central

    Curley, G. F.; Hamid, U. I.; Laffey, J. G.; Abbott, J.; McKenna, D. H.; Fang, X.; Matthay, M. A.; Lee, J. W.

    2014-01-01

    The lack of suitable donors for all solid-organ transplant programs is exacerbated in lung transplantation by the low utilization of potential donor lungs, due primarily to donor lung injury and dysfunction, including pulmonary edema. The current studies were designed to determine if intravenous clinical-grade human mesenchymal stem (stromal) cells (hMSCs) would be effective in restoring alveolar fluid clearance (AFC) in the human ex vivo lung perfusion model, using lungs that had been deemed unsuitable for transplantation and had been subjected to prolonged ischemic time. The human lungs were perfused with 5% albumin in a balanced electrolyte solution and oxygenated with continuous positive airway pressure. Baseline AFC was measured in the control lobe and if AFC was impaired (defined as <10%/h), the lungs received either hMSC (5 × 106 cells) added to the perfusate or perfusion only as a control. AFC was measured in a different lung lobe at 4 h. Intravenous hMSC restored AFC in the injured lungs to a normal level. In contrast, perfusion only did not increase AFC. This positive effect on AFC was reduced by intrabronchial administration of a neutralizing antibody to keratinocyte growth factor (KGF). Thus, intravenous allogeneic hMSCs are effective in restoring the capacity of the alveolar epithelium to remove alveolar fluid at a normal rate, suggesting that this therapy may be effective in enhancing the resolution of pulmonary edema in human lungs deemed clinically unsuitable for transplantation. PMID:24532289

  17. Diagnostic Value of T-cell Interferon-γ Release Assays on Synovial Fluid for Articular Tuberculosis: A Pilot Study

    PubMed Central

    Cheng, Xin-He; Bian, Sai-Nan; Zhang, Yue-Qiu; Zhang, Li-Fan; Shi, Xiao-Chun; Yang, Bo; Zhang, Feng-Chun; Liu, Xiao-Qing

    2016-01-01

    Background: Tuberculosis (TB) remains a major global public health challenge. Articular TB is an important form of extrapulmonary tuberculosis, and its diagnosis is difficult because of the low sensitivity of traditional methods. The aim of this study was to analyze the diagnostic value of T-SPOT.TB on synovial fluid for the diagnosis of articular TB. Methods: Patients with suspected articular TB were enrolled consecutively between August 2011 and December 2015. T-SPOT.TB was performed on both synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs). The final diagnosis of articular TB was independent of the T-SPOT.TB result. The diagnostic sensitivity, specificity, predictive value, and likelihood ratio of T-SPOT.TB on SFMCs and PBMCs were analyzed. Results: Twenty patients with suspected articular TB were enrolled. Six were diagnosed with articular TB, and 14 patients were diagnosed with other diseases. Sensitivity and specificity were 83% and 86% for T-SPOT.TB on SFMCs, and 67% and 69% for T-SPOT.TB on PBMCs, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of T-SPOT.TB on SFMCs were 71% and 92%, respectively. The PPV and NPV were 50% and 82% for T-SPOT.TB on PBMCs. Conclusion: Sensitivity, specificity, and NPV of T-SPOT.TB on SFMCs appeared higher than that on PBMCs, indicating that T-SPOT.TB on SFMCs might be a rapid and accurate diagnostic test for articular TB. PMID:27174325

  18. Oscillatory structural forces due to nonionic surfactant micelles: data by colloidal-probe AFM vs theory.

    PubMed

    Christov, Nikolay C; Danov, Krassimir D; Zeng, Yan; Kralchevsky, Peter A; von Klitzing, Regine

    2010-01-19

    Micellar solutions of nonionic surfactants Brij 35 and Tween 20 are confined between two surfaces in a colloidal-probe atomic-force microscope (CP-AFM). The experimentally detected oscillatory forces due to the layer-by-layer expulsion of the micelles agree very well with the theoretical predictions for hard-sphere fluids. While the experiment gives parts of the stable branches of the force curve, the theoretical model allows reconstruction of the full oscillatory curve. Therewith, the strength and range of the ordering could be determined. The resulting aggregation number from the fits of the force curves for Brij 35 is close to 70 and exhibits a slight tendency to increase with the surfactant concentration. The last layer of micelles cannot be pressed out. The measured force-vs-distance curve has nonequilibrium portions, which represent "jumps" from one to another branch of the respective equilibrium oscillatory curve. In the case of Brij 35, at concentrations <150 mM spherical micelles are present and the oscillation period is close to the micelle diameter, slightly decreasing with the rise of concentration. For elongated micelles (at concentration 200 mM), no harmonic oscillations are observed anymore; instead, the period increases with the decrease of film thickness. In the case of Tween 20, the force oscillations are almost suppressed, which implies that the micelles of this surfactant are labile and are demolished by the hydrodynamic shear stresses due to the colloidal-probe motion. The comparison of the results for the two surfactants demonstrates that in some cases the micelles can be destroyed by the CP-AFM, but in other cases they can be stable and behave as rigid particles. This behavior correlates with the characteristic times of the slow micellar relaxation process for these surfactants. PMID:20067306

  19. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    NASA Astrophysics Data System (ADS)

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-11-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

  20. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    PubMed Central

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-01-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution. PMID:26561004

  1. Bacteria attachment to surfaces--AFM force spectroscopy and physicochemical analyses.

    PubMed

    Harimawan, Ardiyan; Rajasekar, Aruliah; Ting, Yen-Peng

    2011-12-01

    Understanding bacterial adhesion to surfaces requires knowledge of the forces that govern bacterial-surface interactions. Biofilm formation on stainless steel 316 (SS316) by three bacterial species was investigated by examining surface force interaction between the cells and metal surface using atomic force microscopy (AFM). Bacterial-metal adhesion force was quantified at different surface delay time from 0 to 60s using AFM tip coated with three different bacterial species: Gram-negative Massilia timonae and Pseudomonas aeruginosa, and Gram-positive Bacillus subtilis. The results revealed that bacterial adhesion forces on SS316 surface by Gram-negative bacteria is higher (8.53±1.40 nN and 7.88±0.94 nN) when compared to Gram-positive bacteria (1.44±0.21 nN). Physicochemical analysis on bacterial surface properties also revealed that M. timonae and P. aeruginosa showed higher hydrophobicity and surface charges than B. subtilis along with the capability of producing extracellular polymeric substances (EPS). The higher hydrophobicity, surface charges, and greater propensity to form EPS by M. timonae and P. aeruginosa led to high adhesive force on the metal surface. PMID:21889162

  2. AFM Studies of Salt Concentration Effects on the (110) Surface Structure of Tetragonal Lysozyme Crystals

    NASA Technical Reports Server (NTRS)

    Pusey, Marc Lee; Gorti, Sridhar; Forsythe, Elizabeth; Konnert, John

    2002-01-01

    Previous high resolution AFM studies of the (110) surface of tetragonal chicken egg white lysozyme crystals had shown that only one of two possible molecular surfaces is present, those constituting the completed 43 helices. These suggested that the crystal growth process was by the solution-phase assembly of the growth units, which then attach to the surface. However, the best fit for the imaged surfaces, vs. those predicted based upon the bulk crystallographic coordinates, were obtained when the packing about the 43 helices was "tightened up", while maintaining the underlying crystallographic unit cell spacing. This results in a widening of the gap between adjacent helices, and the top- most layer(s) may no longer be in contact. We postulated that the tightened packing about the helices is a result of the high salt concentrations in the bulk solution, used to crystallize the protein, driving hydrophobic interactions. Once the crystal surface is sufficiently buried by subsequent growth layers the ratio of salt to protein molecules decreases and the helices relax to their bulk crystallographic coordinates. The crystal surface helix structure is thus a reflection of the solution structure, and the tightness of the packing about the 43 helices would be a function of the bulk salt concentration. AFM images of the (110) surface of tetragonal lysozyme crystals grown under low (2%) and high (5%) NaCl concentrations reveal differences in the packing about the 43 helices consistent with the above proposal.

  3. Automated AFM force curve analysis for determining elastic modulus of biomaterials and biological samples.

    PubMed

    Chang, Yow-Ren; Raghunathan, Vijay Krishna; Garland, Shaun P; Morgan, Joshua T; Russell, Paul; Murphy, Christopher J

    2014-09-01

    The analysis of atomic force microscopy (AFM) force data requires the selection of a contact point (CP) and is often time consuming and subjective due to influence from intermolecular forces and low signal-to-noise ratios (SNR). In this report, we present an automated algorithm for the selection of CPs in AFM force data and the evaluation of elastic moduli. We propose that CP may be algorithmically easier to detect by identifying a linear elastic indentation region of data (high SNR) rather than the contact point itself (low SNR). Utilizing Hertzian mechanics, the data are fitted for the CP. We first detail the algorithm and then evaluate it on sample polymeric and biological materials. As a demonstration of automation, 64 × 64 force maps were analyzed to yield spatially varying topographical and mechanical information of cells. Finally, we compared manually selected CPs to automatically identified CPs and demonstrated that our automated approach is both accurate (< 10nm difference between manual and automatic) and precise for non-interacting polymeric materials. Our data show that the algorithm is useful for analysis of both biomaterials and biological samples. PMID:24951927

  4. Nanopuller-open data acquisition platform for AFM force spectroscopy experiments.

    PubMed

    Pawlak, Konrad; Strzelecki, Janusz

    2016-05-01

    Atomic Force Microscope (AFM) is a widely used tool in force spectroscopy studies. Presently, this instrument is accessible from numerous vendors, albeit commercial solutions are expensive and almost always hardware and software closed. Approaches for open setups were published, as with modern low cost and readily available piezoelectric actuators, data acquisition interfaces and optoelectronic components building such force spectroscopy AFM is relatively easy. However, suitable software to control such laboratory made instrument was not released. Developing it in the lab requires significant time and effort. Our Nanopuller software described in this paper is intended to eliminate this obstacle. With only minimum adjustments this program can be used to control and acquire data with any suitable National Instruments universal digital/analog interface and piezoelectric actuator analog controller, giving significant freedom and flexibility in designing force spectroscopy experiment. Since the full code, written in a graphical LabVIEW environment is available, our Nanopuller can be easily customized. In this paper we describe the program and test its performance in controlling different setups. Successful and accurate force curve acquisition for standard samples (single molecules of I27O reference titin polyprotein and DNA as well as red blood cells) is shown. PMID:26994468

  5. An approach towards 3D sensitive AFM cantilevers

    NASA Astrophysics Data System (ADS)

    Koops, Richard; Fokkema, Vincent

    2014-04-01

    The atomic force microscope (AFM) tapping mode is a highly sensitive local probing technique that is very useful to study and measure surface properties down to the atomic scale. The tapping mode is mostly implemented using the resonance of the first bending mode of the cantilever and therefore provides sensitivity mainly along the direction of this oscillation. Driven by the semiconductor industry, there is an increasing need for accurate measurements of nanoscale structures for side wall characterization by AFM that requires additional sensitivity in the lateral direction. The conventional tapping mode has been augmented by various authors, for example by tilting the cantilever system (Cho et al 2011 Rev. Sci. Instrum. 82 023707) to access the sidewall or using a torsion mode (Dai et al 2011 Meas. Sci. Technol. 22 094009) of the cantilever to provide additional lateral sensitivity. These approaches however trade lateral sensitivity for vertical sensitivity or still lack sensitivity in the remaining lateral direction. We present an approach towards true 3D sensitivity for AFM cantilevers based on simultaneous excitation and optical detection of multiple cantilever resonance modes along three axes. Tuning the excitation of the cantilever to specific frequencies provides a mechanism to select only those cantilever modes that have the desired characteristics. Additionally, cantilever engineering has been used to design and create a substructure within the cantilever that has been optimized for specific resonance behavior around 4 MHz. In contrast to the conventional approach of using a piezo to actuate the cantilever modulation, we present results on photo-thermal excitation using an intensity modulated low-power laser source. By tightly focusing the excitation spot on the cantilever we were able to attain a deflection efficiency of 0.7 nm µW-1 for the first bending mode. The presented approach results in an efficient all optical excitation and deflection detection

  6. The Advancing State of AF-M315E Technology

    NASA Technical Reports Server (NTRS)

    Masse, Robert; Spores, Ronald A.; McLean, Chris

    2014-01-01

    The culmination of twenty years of applied research in hydroxyl ammonium nitrate (HAN)-based monopropellants, the NASA Space Technology mission Directorate's (STMD) Green Propellant Infusion Mission (GPIM) will achieve the first on-orbit demonstration of an operational AF-M315E green propellant propulsion system by the end of 2015. Following an contextual overview of the completed flight design of the GPIM propellant storage and feed system, results of first operation of a flight-representative heavyweight 20-N engineering model thruster (to be conducted in mid-2014) are presented with performance comparisons to prior lab model (heavyweight) test articles.

  7. Comparison of dynamic lever STM and noncontact AFM

    NASA Astrophysics Data System (ADS)

    Guggisberg, M.; Bammerlin, M.; Lüthi, R.; Loppacher, C.; Battiston, F.; Lü, J.; Baratoff, A.; Meyer, E.; Güntherodt, H.-J.

    We investigate interaction effects which occur in scanning tunneling microscopy (STM) by performing local force spectroscopy with an oscillating tip while imaging Si(111)7×7 terraces in the dynamic lever STM mode (constant time-averaged current). It is found that true atomic resolution is achieved close to the minimum of the resonance frequency vs. distance curve and even closer to the sample. On the other hand true atomic resolution in noncontact AFM (constant frequency shift) is expected several nm away from this minimum, in the range where the frequency shift becomes more negative with decreasing distance.

  8. Characterization of CD30/CD30L+ Cells in Peripheral Blood and Synovial Fluid of Patients with Rheumatoid Arthritis

    PubMed Central

    Dolcino, Marzia; Tinazzi, Elisa; Rigo, Antonella; Argentino, Giuseppe; Patuzzo, Giuseppe; Beri, Ruggero; Puccetti, Antonio

    2015-01-01

    The CD30/CD30L signalling system has been implicated in the pathogenesis of several autoimmune and inflammatory conditions. In rheumatoid arthritis (RA), soluble CD30 (sCD30) levels reflect the recruitment of CD30+ T cells into the inflamed joints and correlate with a positive response to immunosuppressive therapy. The aim of our report was to clarify the role of CD30/CD30L signalling system in the pathogenesis of RA. Our analysis of the CD30L+ T cell subsets in peripheral blood (PB) and synovial fluid (SF) of RA patients and of the related cytokine profiles suggests the involvement of CD30/CD30L signalling in polarization of T cells towards a Th17 phenotype with proinflammatory features. Moreover, in RA SF nearly 50% of Treg cells express CD30, probably as an attempt to downmodulate the ongoing inflammation. We also show here that the engagement of CD30L on neutrophils stimulated with CD30/Fc chimera may play a crucial role in RA inflammation since activated neutrophils release IL-8, thus potentially amplifying the local inflammatory damage. In conclusion, the results obtained suggest that the complex CD30/CD30L signalling pathway is implicated in the pathogenesis and progression of RA synovitis through a concerted action on several immune effector cells. PMID:26090498

  9. Potential role of N-benzylcinnamide in inducing neuronal differentiation from human amniotic fluid mesenchymal stem cells.

    PubMed

    Thangnipon, Wipawan; Puangmalai, Nicha; Suwanna, Nirut; Soi-Ampornkul, Rungtip; Phonchai, Ruchee; Kotchabhakdi, Naiphinich; Mukda, Sujira; Phermthai, Tatsanee; Julavijitphong, Suphakde; Tuchinda, Patoomratana; Nobsathian, Saksit

    2016-01-01

    Neurodegenerative disorders are characterized by chronic and progressive loss of neurons in structure and function related to aging, such as Alzheimer's disease, the latter characterized by the degeneration of cholinergic neurons in basal forebrain connected to the cerebral cortex and hippocampus. Amniotic fluid mesenchymal stem cells (AF-MSCs) have been proposed as one of the candidates for stem cell therapy of nervous system disorders. This study demonstrates that incubation of AF-MSCs, obtained from 16 to 20 week pregnant women, with 10ng/ml bone morphogenetic protein (BMP)-9 for 48h in conditioned medium resulted in transdifferentiation to cholinergic neuronal-like cells. This phenomenon could also be obtained with N-benzylcinnamide (PT-3). Pre-treatment for 1h with 10nM PT-3 augmented BMP-9 transdifferentiation effect, elevated βIII-tubulin cell numbers and fluorescence intensity of immunoreactive ChAT, ameliorated BMP-9-related production of reactive oxygen species and enhanced anti-apoptosis status of the neuronal-like cells. The transdiffirentiation process was accompanied by increased p53 but decreased Notch1 and SIRT1 (p53 deacetylase) levels, and activation of p38, ERK1/2 MAPK, and PI3K/Akt pathways, in concert with inactivation of JNK, all of which were accentuated by PT-3 pre-treatment. These findings suggest that N-benzylcinnamide may provide a useful adjuvant in BMP-9-induced transdifferentiation of AFMSCs into ultimately cholinergic neurons. PMID:26518243

  10. Fluid dilution and efficiency of Na+ transport in a mathematical model of a thick ascending limb cell

    PubMed Central

    Clausen, Chris; Marcano, Mariano; Layton, Anita T.; Layton, Harold E.; Moore, Leon C.

    2013-01-01

    Thick ascending limb (TAL) cells are capable of reducing tubular fluid Na+ concentration to as low as ∼25 mM, and yet they are thought to transport Na+ efficiently owing to passive paracellular Na+ absorption. Transport efficiency in the TAL is of particular importance in the outer medulla where O2 availability is limited by low blood flow. We used a mathematical model of a TAL cell to estimate the efficiency of Na+ transport and to examine how tubular dilution and cell volume regulation influence transport efficiency. The TAL cell model represents 13 major solutes and the associated transporters and channels; model equations are based on mass conservation and electroneutrality constraints. We analyzed TAL transport in cells with conditions relevant to the inner stripe of the outer medulla, the cortico-medullary junction, and the distal cortical TAL. At each location Na+ transport efficiency was computed as functions of changes in luminal NaCl concentration ([NaCl]), [K+], [NH4+], junctional Na+ permeability, and apical K+ permeability. Na+ transport efficiency was calculated as the ratio of total net Na+ transport to transcellular Na+ transport. Transport efficiency is predicted to be highest at the cortico-medullary boundary where the transepithelial Na+ gradient is the smallest. Transport efficiency is lowest in the cortex where luminal [NaCl] approaches static head. PMID:23097469

  11. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta.

    PubMed

    Mareschi, Katia; Castiglia, Sara; Sanavio, Fiorella; Rustichelli, Deborah; Muraro, Michela; Defedele, Davide; Bergallo, Massimiliano; Fagioli, Franca

    2016-02-01

    Mesenchymal stromal cells (MSCs) are a promising tool in cell therapies because of their multipotent, bystander, and immunomodulatory properties. Although bone marrow represents the main source of MSCs, there remains a need to identify a stem cell source that is safe and easily accessible and yields large numbers of cells without provoking debates over ethics. In this study, MSCs isolated from amniotic fluid and placenta were compared with bone marrow MSCs. Their immunomodulatory properties were studied in total activated T cells (peripheral blood mononuclear cells) stimulated with phytohemagglutinin (PHA-PBMCs). In particular, an in vitro co-culture system was established to study: (i) the effect on T-lymphocyte proliferation; (ii) the presence of T regulatory lymphocytes (Treg); (iii) the immunophenotype of various T subsets (Th1 and Th2 naïve, memory, effector lymphocytes); (iv) cytokine release and master gene expression to verify Th1, Th2, and Th17 polarization; and (v) IDO production. Under all co-culture conditions with PHA-PBMCs and MSCs (independently of tissue origin), data revealed: (i) T proliferation inhibition; (ii) increase in naïve T and decrease in memory T cells; (iii) increase in T regulatory lymphocytes; (iv) strong Th2 polarization associated with increased interleukin-10 and interleukin-4 levels, Th1 inhibition (significant decreases in interleukin-2, tumor necrosis factor-α, interferon-γ, and interleukin-12) and Th17 induction (production of high concentrations of interleukins-6 and -17); (v) indoleamine-2,3-dioxygenase mRNA induction in MSCs co-cultured with PHA-PBMCs. AF-MSCs had a more potent immunomodulatory effect on T cells than BM-MSCs, only slightly higher than that of placenta MSCs. This study indicates that MSCs isolated from fetal tissues may be considered a good alternative to BM-MSCs for clinical applications. PMID:26577566

  12. Effect of Varying Fluid Shear Stress on Cancer Stem Cell Viability & Protein Expression

    NASA Astrophysics Data System (ADS)

    Domier, Ria; Kim, Yonghyun; Dozier, David; Triantafillu, Ursula

    2013-11-01

    Cancer stem cells cultured in vitro in stirred bioreactors are exposed to shear stress. By observing the effect of shear stress on cancer stem cell viability, laboratory cell growth could be optimized. In addition, metastasized cancer stem cells in vivo are naturally exposed to shear stress, a factor influencing stem cell differentiation, while circulating in the bloodstream. Changes in protein expression after exposure to shear stress could allow for identification and targeting of circulating cancer cells. In this study, blood flow through capillaries was simulated by using a syringe pump to inject suspensions of Kasumi-1 leukemia stem cells into model blood vessels composed of PEEK tubing 125 microns in diameter. The Hagen-Poisseuille equation was used to solve for operating flow rates based on specified amounts of shear stress. After exposure, cell counts and viabilities were observed using an optical microscope and proteins were analyzed using Western blotting. It was observed that at a one minute exposure to stress, cell viability increased as the amount of shear was increased from 10 to 60 dynes per square centimeter. Results from this research are applicable to optimization of large-scale stem cell growth in bioreactors as well as to the design of targeted cancer therapies. Funding from NSF REU grant #1062611 is gratefully acknowledged.

  13. AFM fluid delivery/liquid extraction surface sampling/electrostatic spray cantilever probe

    SciTech Connect

    Van Berkel, Gary J.

    2015-06-23

    An electrospray system comprises a liquid extraction surface sampling probe. The probe comprises a probe body having a liquid inlet and a liquid outlet, and having a liquid extraction tip. A solvent delivery conduit is provided for receiving solvent liquid from the liquid inlet and delivering the solvent liquid to the liquid extraction tip. An open liquid extraction channel extends across an exterior surface of the probe body from the liquid extraction tip to the liquid outlet. An electrospray emitter tip is in liquid communication with the liquid outlet of the liquid extraction surface sampling probe. A system for analyzing samples, a liquid junction surface sampling system, and a method of analyzing samples are also disclosed.

  14. Scientific management and implementation of the geophysical fluid flow cell for Spacelab missions

    NASA Technical Reports Server (NTRS)

    Hart, J.; Toomre, J.

    1980-01-01

    Scientific support for the spherical convection experiment to be flown on Spacelab 3 was developed. This experiment takes advantage of the zero gravity environment of the orbiting space laboratory to conduct fundamental fluid flow studies concerned with thermally driven motions inside a rotating spherical shell with radial gravity. Such a system is a laboratory analog of large scale atmospheric and solar circulations. The radial body force necessary to model gravity correctly is obtained by using dielectric polarization forces in a radially varying electric field to produce radial accelerations proportional to temperature. This experiment will answer fundamental questions concerned with establishing the preferred modes of large scale motion in planetary and stellar atmospheres.

  15. Mast cell tryptase and carboxypeptidase A expression in body fluid and gastrointestinal tract associated with drug-related fatal anaphylaxis

    PubMed Central

    Guo, Xiang-Jie; Wang, Ying-Yuan; Zhang, Hao-Yue; Jin, Qian-Qian; Gao, Cai-Rong

    2015-01-01

    AIM: To investigate the expression of mast cell tryptase and carboxypeptidase A in drug-related fatal anaphylaxis. METHODS: The expression of mast cell tryptase and carboxypeptidase A in 15 autopsy cases of drug-related fatal anaphylaxis and 20 normal autopsy cases were detected. First, the expression of mast cell tryptase was determined in stomach, jejunum, lung, heart, and larynx by immunofluorescence. Different tissues were removed and fixed in paraformaldehyde solution, then paraffin sections were prepared for immunofluorescence. Using specific mast cell tryptase and carboxypeptidase A antibodies, the expression of tryptase and carboxypeptidase A in gastroenterology tract and other tissues were observed using fluorescent microscopy. The postmortem serum and pericardial fluid were collected from drug-related fatal anaphylaxis and normal autopsy cases. The level of mast cell tryptase and carboxypeptidase A in postmortem serum and pericardial fluid were measured using fluor enzyme linked immunosorbent assay (FEIA) and enzyme linked immunosorbent assay (ELISA) assay. The expression of mast cell tryptase and carboxypeptidase A was analyzed in drug-related fatal anaphylaxis cases and compared to normal autopsy cases. RESULTS: The expression of carboxypeptidase A was less in the gastroenterology tract and other tissues from anaphylaxis-related death cadavers than normal controls. Immunofluorescence revealed that tryptase expression was significantly increased in multiple organs, especially the gastrointestinal tract, from anaphylaxis-related death cadavers compared to normal autopsy cases (46.67 ± 11.11 vs 4.88 ± 1.56 in stomach, 48.89 ± 11.02 vs 5.21 ± 1.34 in jejunum, 33.72 ± 5.76 vs 1.30 ± 1.02 in lung, 40.08 ± 7.56 vs 1.67 ± 1.03 in larynx, 7.11 ± 5.67 vs 1.10 ± 0.77 in heart, P < 0.05). Tryptase levels, as measured with FEIA, were significantly increased in both sera (43.50 ± 0.48 μg/L vs 5.40 ± 0.36 μg/L, P < 0.05) and pericardial fluid (28.64 ± 0

  16. The differentiation of amniotic fluid stem cells into sweat glandlike cells is enhanced by the presence of Sonic hedgehog in the conditioned medium.

    PubMed

    Liang, Hansi; Sun, Qing; Zhen, Yunfang; Li, Fang; Xu, YunYun; Liu, Yao; Zhang, Xueguang; Qin, Mingde

    2016-09-01

    After patients suffer severe full-thickness burn injuries, the current treatments cannot lead to the complete self-regeneration of the sweat gland structure and function. Therefore, it is important to identify new methods for acquiring sufficient functional sweat gland cells to restore skin function. In this study, we induced CD117+ human amniotic fluid stem (hAFS) cells to differentiate into sweat glandlike (hAFS-SG) cells based on the use of conditioned medium (CM) from the human sweat gland (hSG) cells. Real-time PCR and immunofluorescent staining were used to confirm the expression of the sweat gland-related genes Ectodysplasin-A (EDA), Ectodysplasin-A receptor (EDAR), keratin 8 (K8) and carcino-embryonic antigen (CEA). Transmission electron microscopy analysis shows that microvilli, the cellular structures that are typical for hSG cells, can also be observed on the membrane of the hAFS-SG cells. Our test for the calcium response to acetylcholine (Ach) proved that hAFS-SG cells have the potential to respond to Ach in a manner similar to normal sweat glands. A three-dimensional culture is an effective approach that stimulates the hAFS-SG cells to form tubular structures and drives hAFS-SG cells to mature into higher stage. We also found that epidermal growth factor enhances the efficiency of differentiation and that Sonic hedgehog is an important factor of the CM that influences sweat gland differentiation. Our study provides the basis for further investigations into novel methods of inducing stem cells to differentiate into sweat glandlike cells. PMID:27120089

  17. Anterior horn cell loss from subdural hygroma: a consequence of spontaneous spinal fluid leak.

    PubMed

    Mihaylova, Temenuzhka; Biondo, Andrew; Zak, Imad; Lewis, Richard A

    2011-06-15

    We describe a case of a 50-year-old man with bilateral shoulder girdle weakness caused by anterior subdural hygroma secondary to a previous spontaneous CSF leak. The CSF leak occurred and resolved 16 years prior to presenting with a 6-year progressive painless, asymmetric proximal muscle weakness involving both upper extremities. Current examination reveals remarkably restricted atrophy and weakness in bilateral C5-6 muscles and absent biceps and brachioradialis reflexes. Neuroimaging shows a subdural CSF collection extending from C1 to L2 anteriorly causing thecal sac effacement at the C4 level and secondary Chiari deformity. The clinical picture demonstrates severe weakness in C5-6 muscles with sparing of all other myotomes. The acute clinical features as well as neuroimaging characteristics of spontaneous CSF leak are well known but the late effects are less described. The development of a subdural fluid collection secondary to a spinal fluid leak can cause damage to the anterior spinal cord years after the leak. The underlying pathophysiology of the motor neuron loss remains unclear but there appears to be a pressure effect localized to the C4-5 region. The possibility that intervention to prevent or treat the subdural CSF collection might have avoided the shoulder girdle weakness is considered. PMID:21440260

  18. Cerebrospinal Fluid (CSF) CD8+ T-Cells That Express Interferon-Gamma Contribute to HIV Associated Neurocognitive Disorders (HAND)

    PubMed Central

    Schrier, Rachel D.; Hong, Suzi; Crescini, Melanie; Ellis, Ronald; Pérez-Santiago, Josué; Spina, Celsa; Letendre, Scott

    2015-01-01

    Background HIV associated neurocognitive disorders (HAND) continue to affect cognition and everyday functioning despite anti-retroviral treatment (ART). Previous studies focused on mechanisms related to monocyte/macrophage mediated inflammation. However, in the ART era, there is increasing evidence for the involvement of CD8+ T-cells in CNS pathogenesis. Methods To investigate the relationship between T-cell responses and neurocognitive impairment (NCI), cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T-cell intracellular cytokine (IFNγ, IL-2, TNFα) and lytic marker (CD107a) expression were assessed in HIV infected subjects who underwent comprehensive neurocognitive (NC) evaluation and either initiated or changed ART. Results Data were collected from 31 participants at 70 visits. The frequency of cytokine expressing T-cells in CSF was significantly higher than in peripheral blood for CD4+T-cells: TNFα, IL-2, IFNγ and CD8+T-cells: IL-2 and IFNγ. Analysis of T-cell activity and NCI as a function of CSF HIV RNA levels suggested a general association between NCI, high CSF CD8+ (but not CD4+T-cell) cytokine expression and CSF HIV RNA <103 copies/ml (p<0.0001). Specifically, CSF CD8+ T-cell IFNγ expression correlated with severity of NCI (r = 0.57, p = 0.004). Multivariable analyses indicated that CSF CD8+T-cell IFNγ and myeloid activation (CD163) contributed equally and independently to cognitive status and a composite variable produced the strongest correlation with NCI (r = 0.83, p = 0.0001). In contrast, CD8+ cytolytic activity (CD107a expression) was negatively correlated with NCI (p = 0.05) but was dependent on CD4 levels >400/μl and low CSF HIV RNA levels (<103 copies/ml). In our longitudinal analysis of 16 subjects, higher CSF CD8+IFNγ expression at baseline predicted NC decline at follow-up (p = 0.02). Severity of NCI at follow-up correlated with level of residual HIV RNA in CSF. Conclusions Presence of IFNγ expressing CD8+ T-cells

  19. Therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia using Fe2O3 nanoparticles

    PubMed Central

    Yan, S.Y.; Chen, M.M.; Fan, J.G.; Wang, Y.Q.; Du, Y.Q.; Hu, Y.; Xu, L.M.

    2014-01-01

    This study aimed to investigate the therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia (MFH) using Fe2O3 nanoparticles. Hepatocarcinoma SMMC-7721 cells cultured in vitro were treated with ferrofluid containing Fe2O3 nanoparticles and irradiated with an alternating radio frequency magnetic field. The influence of the treatment on the cells was examined by inverted microscopy, MTT and flow cytometry. To study the therapeutic mechanism of the Fe2O3 MFH, Hsp70, Bax, Bcl-2 and p53 were detected by immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). It was shown that Fe2O3 MFH could cause cellular necrosis, induce cellular apoptosis, and significantly inhibit cellular growth, all of which appeared to be dependent on the concentration of the Fe2O3 nanoparticles. Immunocytochemistry results showed that MFH could induce high expression of Hsp70 and Bax, decrease the expression of mutant p53, and had little effect on Bcl-2. RT-PCR indicated that Hsp70 expression was high in the early stage of MFH (<24 h) and became low or absent after 24 h of MFH treatment. It can be concluded that Fe2O3 MFH significantly inhibited the proliferation of in vitro cultured liver cancer cells (SMMC-7721), induced cell apoptosis and arrested the cell cycle at the G2/M phase. Fe2O3 MFH can induce high Hsp70 expression at an early stage, enhance the expression of Bax, and decrease the expression of mutant p53, which promotes the apoptosis of tumor cells. PMID:25296356

  20. Therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia using Fe₂O₃ nanoparticles.

    PubMed

    Yan, S Y; Chen, M M; Fan, J G; Wang, Y Q; Du, Y Q; Hu, Y; Xu, L M

    2014-11-01

    This study aimed to investigate the therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia (MFH) using Fe₂O₃ nanoparticles. Hepatocarcinoma SMMC-7721 cells cultured in vitro were treated with ferrofluid containing Fe₂O₃ nanoparticles and irradiated with an alternating radio frequency magnetic field. The influence of the treatment on the cells was examined by inverted microscopy, MTT and flow cytometry. To study the therapeutic mechanism of the Fe₂O₃ MFH, Hsp70, Bax, Bcl-2 and p53 were detected by immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). It was shown that Fe₂O₃ MFH could cause cellular necrosis, induce cellular apoptosis, and significantly inhibit cellular growth, all of which appeared to be dependent on the concentration of the Fe₂O₃nanoparticles. Immunocytochemistry results showed that MFH could induce high expression of Hsp70 and Bax, decrease the expression of mutant p53, and had little effect on Bcl-2. RT-PCR indicated that Hsp70 expression was high in the early stage of MFH (<24 h) and became low or absent after 24 h of MFH treatment. It can be concluded that Fe₂O₃MFH significantly inhibited the proliferation of in vitro cultured liver cancer cells (SMMC-7721), induced cell apoptosis and arrested the cell cycle at the G₂/M phase. Fe₂O₃ MFH can induce high Hsp70 expression at an early stage, enhance the expression of Bax, and decrease the expression of mutant p53, which promotes the apoptosis of tumor cells. PMID:25296356

  1. Therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia using Fe2O3 nanoparticles.

    PubMed

    Yan, S Y; Chen, M M; Fan, J G; Wang, Y Q; Du, Y Q; Hu, Y; Xu, L M

    2014-08-29

    This study aimed to investigate the therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia (MFH) using Fe2O3 nanoparticles. Hepatocarcinoma SMMC-7721 cells cultured in vitro were treated with ferrofluid containing Fe2O3 nanoparticles and irradiated with an alternating radio frequency magnetic field. The influence of the treatment on the cells was examined by inverted microscopy, MTT and flow cytometry. To study the therapeutic mechanism of the Fe2O3 MFH, Hsp70, Bax, Bcl-2 and p53 were detected by immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). It was shown that Fe2O3 MFH could cause cellular necrosis, induce cellular apoptosis, and significantly inhibit cellular growth, all of which appeared to be dependent on the concentration of the Fe2O3 nanoparticles. Immunocytochemistry results showed that MFH could induce high expression of Hsp70 and Bax, decrease the expression of mutant p53, and had little effect on Bcl-2. RT-PCR indicated that Hsp70 expression was high in the early stage of MFH (<24 h) and became low or absent after 24 h of MFH treatment. It can be concluded that Fe2O3 MFH significantly inhibited the proliferation of in vitro cultured liver cancer cells (SMMC-7721), induced cell apoptosis and arrested the cell cycle at the G2/M phase. Fe2O3 MFH can induce high Hsp70 expression at an early stage, enhance the expression of Bax, and decrease the expression of mutant p53, which promotes the apoptosis of tumor cells. PMID:25184378

  2. Physical properties of polyacrylamide gels probed by AFM and rheology

    NASA Astrophysics Data System (ADS)

    Abidine, Yara; Laurent, Valérie M.; Michel, Richard; Duperray, Alain; Iulian Palade, Liviu; Verdier, Claude

    2015-02-01

    Polymer gels have been shown to behave as viscoelastic materials but only a small amount of data is usually provided in the glass transition. In this paper, the dynamic moduli G\\prime and G\\prime\\prime of polyacrylamide hydrogels are investigated using both an AFM in contact force modulation mode and a classical rheometer. The validity is shown by the matching of the two techniques. Measurements are carried out on gels of increasing polymer concentration in a wide frequency range. A model based on fractional derivatives is successfully used, covering the whole frequency range. G\\text{N}0 , the plateau modulus, as well as several other parameters are obtained at low frequencies. The model also predicts the slope a of both moduli in the glass transition, and a transition frequency f\\text{T} is introduced to separate the gel-like behavior with the glassy state. Its variation with polymer content c gives a dependence f\\text{T}∼ c1.6 , in good agreement with previous theories. Therefore, the AFM data provides new information on the physics of polymer gels.

  3. Dual AFM probes alignment based on vision guidance

    NASA Astrophysics Data System (ADS)

    Zhang, Hua-kun; Gao, Si-tian; Lu, Ming-zhen; Wang, Long-long

    2013-10-01

    Atomic force microscope (AFM) with dual probes that operate together can measure both side walls excellently at the same time, which virtually eliminates the prevalent effect of probe width that contributes a large component of uncertainty in measurement results and finally obtains the critical dimension (CD)(e.g. the linewidth) through data synthesis. In calibration process, the dual probes must contact each other in advance, which realizes the alignment in the three dimensions, to establish a zero reference point and ensure the accuracy of measurement. Because nowadays the optical resolution of advanced lens have exceeded micrometer range, and the size of probes is within micro level, it is possible to acquire dual probes images in both horizontal and vertical directions, through which the movement of the probes can be controlled in time. In order to further enhance the alignment precision, sub-pixel edge detection method based on Zernike orthogonal moment is used to obtain relative position between these two probes, which helps the tips alignment attains sub-micron range. Piezoelectric nanopositioning stages calibrated by laser interferometer are used to implement fine movement of the probes to verify the accuracy of the experimental results. To simplify the system, novel self-sensing and self-actuating probe based on a quartz tuning fork combined with a micromachined cantilever is used for dynamic mode AFM. In this case, an external optical detection system is not needed, so the system is simple and small.

  4. Viscoelasticity of gelatin surfaces probed by AFM noise analysis.

    PubMed

    Benmouna, Farida; Johannsmann, Diethelm

    2004-01-01

    The viscoelastic properties of surfaces of swollen gelatin were investigated by analyzing the Brownian motion of an atomic force microscopy (AFM) cantilever in contact with the gel surface. A micron-sized glass sphere attached to the AFM cantilever is used as the dynamic probe. When the sphere approaches the gelatin surface, there is a static repulsive force without a jump into contact. The cantilever's Brownian movement is monitored in parallel, providing access to the dynamic sphere-surface interaction as quantified by the dynamic spring constant, kappa, and the drag coefficient, xi. Gelatin is used as a model substance for a variety of other soft surfaces, where the stiffness of the gel can be varied via the solvent quality, the bloom number, and the pH. The modulus derived from the static force-distance curve is in the kPa range, consistent with the literature. However, the dynamic spring constant as derived from the Brownian motion is much larger than the static differential spring constant dF/dz. On retraction, one observes a rather strong adhesion hysteresis. The strength of the bridge (as given by the dynamic spring constant and the drag coefficient) is very small. PMID:15745019

  5. Analysis of AFM cantilever dynamics close to sample surface

    NASA Astrophysics Data System (ADS)

    Habibnejad Korayem, A.; Habibnejad Korayem, Moharam; Ghaderi, Reza

    2013-07-01

    For imaging and manipulation of biological specimens application of atomic force microscopy (AFM) in liquid is necessary. In this paper, tapping-mode AFM cantilever dynamics in liquid close to sample surface is modeled and simulated by well defining the contact forces. The effect of cantilever tilting angle has been accounted carefully. Contact forces have some differences in liquid in comparison to air or vacuum in magnitude or formulation. Hydrodynamic forces are also applied on the cantilever due to the motion in liquid. A continuous beam model is used with its first mode and forward-time simulation method for simulation of its hybrid dynamics and the frequency response and amplitude versus separation diagrams are extracted. The simulation results show a good agreement with experimental results. The resonance frequency in liquid is so small in comparison to air due to additional mass and also additional damping due to the viscosity of the liquid around. The results show that the effect of separation on free vibration amplitude is great. Its effect on resonance frequency is considerable too.

  6. Iron oxide mineral-water interface reactions studied by AFM

    SciTech Connect

    Hawley, M.E.; Rogers, P.S.Z.

    1994-07-01

    Natural iron mineral surfaces have been examined in air by atomic force (AFM) and scanning tunneling (STM) microscopies. A number of different surface features were found to be characteristic of the native surface. Even surfaces freshly exposed by crushing larger crystals were found to have a pebbly surface texture caused by the presence of thin coatings of what might be surface precipitates. This finding is interpreted as evidence for previous exposure to water, probably through an extensive network of microfractures. Surface reactions on the goethite crystals were studied by AFM at size resolutions ranging from microns to atomic resolution before, during, and after reaction with distilled water and 0.lN HCl. Immediate and extensive surface reconfiguration occurred on contact with water. In one case, after equilibration with water for 3 days, surface reprecipitation, etching and pitting were observed. Atomic resolution images taken under water were found to be disordered. The result of surface reaction was generally to increase the surface area substantially through the extension of surface platelet arrays, present prior to reaction. This work is being done in support of the site characterization project at Yucca Mountain.

  7. Pathogen identification using peptide nanotube biosensors and impedance AFM

    NASA Astrophysics Data System (ADS)

    Maccuspie, Robert I.

    Pathogen identification at highly sensitive levels is crucial to meet urgent needs in fighting the spread of disease or detecting bioterrorism events. Toward that end, a new method for biosensing utilizing fluorescent antibody nanotubes is proposed. Fundamental studies on the self-assembly of these peptide nanotubes are performed, as are applications of aligning these nanotubes on surfaces. As biosensors, these nanotubes incorporate recognition units with antibodies at their ends and fluorescent signaling units at their sidewalls. When viral pathogens were mixed with these antibody nanotubes in solution, the nanotubes rapidly aggregated around the viruses. The size of the aggregates increased as the concentration of viruses increased, as detected by flow cytometry on the order of attomolar concentrations by changes in fluorescence and light scattering intensities. This enabled determination of the concentrations of viruses at trace levels (102 to 106 pfu/mL) within 30 minutes from the receipt of samples to the final quantitative data analysis, as demonstrated on Adenovirus, Herpes Simplex Virus, Influenza, and Vaccinia virus. As another separate approach, impedance AFM is used to study the electrical properties of individual viruses and nanoparticles used as model systems. The design, development, and implementation of the impedance AFM for an Asylum Research platform is described, as well as its application towards studying the impedance of individual nanoparticles as a model system for understanding the fundamental science of how the life cycle of a virus affects its electrical properties. In combination, these approaches fill a pressing need to quantify viruses both rapidly and sensitively.

  8. Comparison of particle sizes determined with impactor, AFM and SEM

    NASA Astrophysics Data System (ADS)

    Gwaze, Patience; Annegarn, Harold J.; Huth, Joachim; Helas, Günter

    2007-11-01

    Particles size comparisons were made between conventional aerodynamic and mobility sizing techniques and physical geometric sizes measured by high resolution microscopes. Atmospheric particles were collected during the wet and dry seasons in the Amazonian ecosystems. Individual particles deposited on four stages of the MOUDI (Micro-Orifice Uniform Deposition Impactor) were characterised for particle volumes, projected surface diameters and morphologies with an Atomic Force Microscope (AFM) and a Scanning Electron Microscope (SEM). AFM and SEM size distributions were verified against distributions derived from response functions of individual MOUDI stages as specified by Winklmayr et al. [Winklmayr, W., Wang, H.-C., John, W., 1990. Adaptation of the Twomey algorithm to the inversion of cascade impactor data. Aerosol Science and Technology 13, 322-331.]. Particles indicated inherent discrepancies in sizing techniques. Particle volumes were systematically lower than expected by factors of up to 3.6. Differences were attributed to loss of mass, presumably water adsorbed on particles. Losses were high and could not be accounted for by measured humidity growth factors suggesting significant losses of other volatile compounds as well, particularly on particles that were collected during the wet season. Microscopy results showed that for hygroscopic particles, microscopy sizes depend on the relative humidity history of particles before and after sampling. Changes in relative humidity significantly altered particle morphologies. Depending on when changes occur, such losses will bias not only microscopy particle sizes but also impactor mass distributions and number concentrations derived from collected particles.

  9. AFM analysis of bleaching effects on dental enamel microtopography

    NASA Astrophysics Data System (ADS)

    Pedreira de Freitas, Ana Carolina; Espejo, Luciana Cardoso; Botta, Sergio Brossi; Teixeira, Fernanda de Sa; Luz, Maria Aparecida A. Cerqueira; Garone-Netto, Narciso; Matos, Adriana Bona; Salvadori, Maria Cecilia Barbosa da Silveira

    2010-02-01

    The purpose of this in vitro study was to test a new methodology to evaluate the effects of 35% hydrogen peroxide agent on the microtopography of sound enamel using an atomic force microscope (AFM). The buccal sound surfaces of three extracted human lower incisors were used, without polishing the surfaces to maintain them with natural morphology. These unpolished surfaces were subjected to bleaching procedure with 35% hydrogen peroxide that consisted of 4 applications of the bleaching agent on enamel surfaces for 10 min each application. Surface images were obtained in a 15 μm × 15 μm area using an AFM. The roughness (Ra and RMS) and the power spectral density (PSD) were obtained before and after the bleaching treatment. As results we could inquire that the PSD analyses were very suitable to identifying the morphological changes on the surfaces, while the Ra and RMS parameters were insufficient to represent the morphological alterations promoted by bleaching procedure on enamel. The morphological wavelength in the range of visible light spectrum (380-750 nm) was analyzed, showing a considerable increase of the PSD with the bleaching treatment.

  10. Clone-derived human AF-amniotic fluid stem cells are capable of skeletal myogenic differentiation in vitro and in vivo.

    PubMed

    Ma, Xiaorong; Zhang, Shengli; Zhou, Junmei; Chen, Baisong; Shang, Yafeng; Gao, Tongbing; Wang, Xue; Xie, Hua; Chen, Fang

    2012-08-01

    Stem cell-based therapy may be the most promising method to cure skeletal muscle degenerative diseases such as Duchenne muscular dystrophy (DMD) and trauma in the future. Human amniotic fluid is enriched with early-stage stem cells from developing fetuses and these cells have cardiomyogenic potential both in vitro and in vivo. In the present study, we investigated the characteristics of human amniotic fluid-derived AF-type stem (HAF-AFS) cells by flow cytometry, immunofluorescence staining, reverse-transcription polymerase chain reaction, and osteogenic and adipogenic differentiation analysis. After confirming the stemness of HAF-AFS cells, we tested whether HAF-AFS cells could differentiate into skeletal myogenic cells in vitro and incorporate into regenerating skeletal muscle in vivo. By temporary exposure to the DNA demethylation agent 5-aza-2'-deoxycytidine (5-Aza dC) or co-cultured with C2C12 myoblasts, HAF-AFS cells differentiated into skeletal myogenic cells, expressing skeletal myogenic cell-specific markers such as Desmin, Troponin I (Tn I) and α-Actinin. Four weeks after transplantation into cardiotoxin-injured and X-ray-irradiated tibialis anterior (TA) muscles of NOD/SCID mice, HAF-AFS cells survived, differentiated into myogenic precursor cells and fused with host myofibres. The findings that HAF-AFS cells differentiate into myogenic cells in vitro and incorporate in skeletal muscle regeneration in vivo hold the promise of HAF-AFS cell-based therapy for skeletal muscle degenerative diseases. PMID:22396316

  11. Enhancement of Anti-Hypoxic Activity and Differentiation of Cardiac Stem Cells by Supernatant Fluids from Cultured Macrophages that Phagocytized Dead Mesenchymal Stem Cells

    PubMed Central

    Liu, Liang; Jin, Xian; Zhou, Zhong’e; Shen, Chengxing

    2016-01-01

    Background: Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown. Methods: MSCs, CSCs, and macrophages were obtained from bone marrow, hearts, and peritoneal cavity of mice, respectively. dMSCs were harvested after hypoxia for 24 h, and incubated with macrophages (2:1) for another 2 days with or without lipopolysaccharide (LPS, 50 ng/mL) and sorted by flow cytometry to obtain pMΦ. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Expression of inflammation and growth factors was assessed by real-time polymerase chain reaction (RT-PCR) in MΦ, LPS-pMΦ, and pMΦ cells. Results: pMΦ expressed higher levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)and lower levels of tumor necrosis factor-α(TNF-α)and IL-6 than LPS-pMΦ, higher levels of growth factors and of GATA-4 and c-TnI at the 7th day, which were similar to those in MSCs. CSCs cultured with supernatant fluids of pMΦ exhibited higher proliferative, anti-hypoxic, and differentiation activities. Conclusion: The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Furthermore, some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of

  12. A cryogenic high pressure cell for inelastic neutron scattering measurements of quantum fluids and solids

    SciTech Connect

    Carmichael, Justin R; Omar Diallo, Souleymane

    2013-01-01

    We present our new development of a high pressure cell for inelastic neutron scattering measurements of helium at ultra-low temperatures. The cell has a large sample volume of ~140 cm3, and a working pressure of ~70 bar, with a relatively thin wall-thickness (1.1 mm) - thanks to the high yield strength aluminum used in the design. Two variants of this cell have been developed; one with permanently joined components using electron-beam welding and explosion welding, methods that have little or no impact on the global heat treatment of the cell, and another with modular and interchangeable components, which include a capacitance pressure gauge, that can be sealed using traditional indium wire technique. The performance of the cell has been tested in recent measurements on superfluid liquid helium near the solidification line.

  13. Acute and chronic wound fluids inversely influence adipose-derived stem cell function: molecular insights into impaired wound healing.

    PubMed

    Koenen, Paola; Spanholtz, Timo A; Maegele, Marc; Stürmer, Ewa; Brockamp, Thomas; Neugebauer, Edmund; Thamm, Oliver C

    2015-02-01

    Wound healing is a complex biological process that requires a well-orchestrated interaction of mediators as well as resident and infiltrating cells. In this context, mesenchymal stem cells play a crucial role as they are attracted to the wound site and influence tissue regeneration by various mechanisms. In chronic wounds, these processes are disturbed. In a comparative approach, adipose-derived stem cells (ASC) were treated with acute and chronic wound fluids (AWF and CWF, respectively). Proliferation and migration were investigated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and transwell migration assay. Gene expression changes were analysed using quantitative real time-polymerase chain reaction. AWF had a significantly stronger chemotactic impact on ASC than CWF (77·5% versus 59·8% migrated cells). While proliferation was stimulated by AWF up to 136·3%, CWF had a negative effect on proliferation over time (80·3%). Expression of b-FGF, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 was strongly induced by CWF compared with a mild induction by AWF. These results give an insight into impaired ASC function in chronic wounds. The detected effect of CWF on proliferation and migration of ASC might be one reason for an insufficient healing process in chronic wounds. PMID:23490259

  14. Implementation of a four quadrant optic fibre bundle as a deflection sensor to get rid of heat sources in an AFM head

    NASA Astrophysics Data System (ADS)

    Boukellal, Younes; Ducourtieux, Sebastien

    2015-09-01

    In the frame of developing a thermally passive atomic force microscope head, a new kind of 2D displacement sensor based on a four quadrant optic fibre bundle has been implemented. The aim is to replace the quad cell photodiode used in the optical beam deflection method to detect cantilever deflection. The use of the bundle as a position sensor has already been modelled and experimentally evaluated in a previous work. This article reports on the implementation of the bundle as a deflection sensor for atomic force microscopy. The main motivation for such a development was to reduce the heat sources in the instrument. To reach this goal the photodiode and its conditioning circuit used for the measurement of cantilever deflection has been externalized from the AFM head. For the same reason, the laser diode and its electronic driver have been deported using optic fibre. To test the AFM head prototype in real conditions, approach curves and AFM images have been performed. The results show that the bundle is very well suited for AFM applications that require very low heat sources such as metrological AFM where each error source has to be managed.

  15. Fuel cell assembly fluid flow plate having conductive fibers and rigidizing material therein

    DOEpatents

    Walsh, Michael M.

    2000-01-01

    A fluid flow plate is preferably formed with three initial sections, for instance, two layers of conductive (e.g., metal) fibers and a barrier material (e.g., metal foil) which is interposed between the two layers. For example, sintering of these three sections can provide electrical path(s) between outer faces of the two layers. Then, the sintered sections can be, for instance, placed in a mold for forming of flow channel(s) into one or more of the outer faces. Next, rigidizing material (e.g., resin) can be injected into the mold, for example, to fill and/or seal space(s) about a conductive matrix of the electrical path(s). Preferably, abrading of surface(s) of the outer face(s) serves to expose electrical contact(s) to the electrical path(s).

  16. Experiments on Thermal Convection in Rotating Spherical Shells With Radial Gravity: The Geophysical Fluid Flow Cell

    NASA Technical Reports Server (NTRS)

    Hart, John E.

    1996-01-01

    Experiments designed to study the fluid dynamics of buoyancy driven circulations in rotating spherical shells were conducted on the United States Microgravity Laboratory 2 spacelab mission. These experiments address several aspects of prototypical global convection relevant to large scale motions on the Sun, Earth, and on the giant planets. The key feature is the consistent modeling of radially directed gravity in spherical geometry by using dielectric polarization forces. Imagery of the planforms of thermally driven flows for rapidly-rotating regimes shows an initial separation and eventual merger of equatorial and polar convection as the heating (i.e. the Rayleigh number) is increased. At low rotation rates, multiple-states of motion for the same external parameters were observed.

  17. FGF2 plays a key role in embryonic cerebrospinal fluid trophic properties over chick embryo neuroepithelial stem cells.

    PubMed

    Martín, C; Bueno, D; Alonso, M I; Moro, J A; Callejo, S; Parada, C; Martín, P; Carnicero, E; Gato, A

    2006-09-15

    During early stages of brain development, neuroepithelial stem cells undergo intense proliferation as neurogenesis begins. Fibroblast growth factor 2 (FGF2) has been involved in the regulation of these processes, and although it has been suggested that they work in an autocrine-paracrine mode, there is no general agreement on this because the behavior of neuroepithelial cells is not self-sufficient in explants cultured in vitro. In this work, we show that during early stages of development in chick embryos there is another source of FGF2, besides that of the neuroepithelium, which affects the brain primordium, since the cerebrospinal fluid (E-CSF) contains several isoforms of this factor. We also demonstrate, both in vitro and in vivo, that the FGF2 from the E-CSF has an effect on the regulation of neuroepithelial cell behavior, including cell proliferation and neurogenesis. In order to clarify putative sources of FGF2 in embryonic tissues, we detected by in situ hybridization high levels of mRNA expression in notochord, mesonephros and hepatic primordia, and low levels in brain neuroectoderm, corroborated by semiquantitative PCR analysis. Furthermore, we show that the notochord segregates several FGF2 isoforms which modify the behavior of the neuroepithelial cells in vitro. In addition, we show that the FGF2 ligand is present in the embryonic serum; and, by means of labeled FGF2, we prove that this factor passes via the neuroepithelium from the embryonic serum to the E-CSF in vivo. Considering all these results, we propose that, in chick embryos, the behavior of brain neuroepithelial stem cells at the earliest stages of development is influenced by the action of the FGF2 contained within the E-CSF which could have an extraneural origin, thus suggesting a new and complementary way of regulating brain development. PMID:16916506

  18. Effect of oral N-acetylcysteine (NAC) on volume and albumin content of respiratory tract fluid but not on epithelial secretory cell number in "smoking" rats.

    PubMed

    Robinson, N; Brattsand, R; Dahlbäck, M

    1990-03-01

    This study was designed to look at the effect of N-acetylcysteine (NAC) on epithelial secretory cells and the respiratory tract fluid volume and albumin content from the lower airways of "bronchitic" rats. Rats were exposed either to tobacco smoke (TS), TS and NAC, or NAC alone. TS caused a significant increase in epithelial secretory cell number which was not reduced by concomitant NAC administration; NAC alone had no effect on cell numbers. TS increased respiratory tract fluid volume and albumin content by a small but non-significant amount, whereas TS and NAC increased the volume and albumin content by a greater and significant amount; NAC alone was also shown to significantly increase both fluid volume and albumin content. PMID:2340888

  19. Agreement of manual cell counts and automated counts of the scil Vet abc Plus(+) hematology analyzer for analysis of equine synovial fluid.

    PubMed

    Van de Water, Eline; Oosterlinck, Maarten; Duchateau, Luc; Pille, Frederik

    2016-06-01

    The purpose of this study was to determine whether the scil Vet abc Plus(+) (SCIL Animal Care Company, Altorf, France), an impedance hematology analyzer, can accurately quantify and differentiate nucleated blood cells (NBCs) in equine synovial fluid. Synovial fluid samples (n=242) in different stages of experimentally induced inflammation were analyzed with and without hyaluronidase pretreatment and compared to manual hemocytometer counts and smear reviews. No significant effect of hyaluronidase pretreatment was observed. Total nucleated cell counts of the scil Vet abc Plus(+) were significantly higher compared to the manual method (P=0.02), yet the difference was small and clinically irrelevant (ratio manual/automated count equal to 0.97 with 95% CI [0.95, 1.00]). Differential cell counts of the scil Vet abc Plus(+) were not accurate. In conclusion, the scil Vet abc Plus(+) hematology analyzer is highly accurate for quantification, but not accurate for differentiation of NBCs in equine synovial fluid. PMID:27234537

  20. Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu-3

    PubMed Central

    Huang, Junwei; Shan, Jiajie; Kim, Dusik; Liao, Jie; Evagelidis, Alexandra; Alper, Seth L; Hanrahan, John W

    2012-01-01

    Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl−/HCO3− exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short-circuit current (Isc). We have studied the role of AE2 in Cl− and fluid secretion by the airway epithelial cell line Calu-3. After confirming expression of its mRNA and protein, a knock-down cell line called AE2-KD was generated by lentivirus-mediated RNA interference in which AE2 mRNA and protein levels were reduced ≥90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na+–K+–2Cl− cotransporter) or NBCe1 (Na+–nHCO3− cotransporter). cAMP agonists stimulated fluid secretion by parental Calu-3 and scrambled shRNA cells >6.5-fold. In AE2-KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO3−] as compared with the control lines. Unstimulated equivalent short-circuit current (Ieq) was elevated in AE2-KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide-induced reductions in both Ieq and fluid secretion were more pronounced in AE2-KD cells. Basolateral Cl−/HCO3− exchange measured by basolateral pH-stat in cells with permeabilized apical membranes was abolished in AE2-KD monolayers, and the intracellular alkalinization resulting from basolateral Cl− removal was reduced by ∼80% in AE2-KD cells. These results identify AE2 as a major pathway for basolateral Cl− loading during cAMP-stimulated secretion of Cl− and fluid by Calu-3 cells, and help explain the large bumetanide-insensitive component of fluid secretion reported previously in airway

  1. Comparative Analysis of Cell-Associated HIV DNA Levels in Cerebrospinal Fluid and Peripheral Blood by Droplet Digital PCR

    PubMed Central

    de Oliveira, Michelli Faria; Gianella, Sara; Letendre, Scott; Scheffler, Konrad; Kosakovsky Pond, Sergei L.; Smith, Davey M.; Strain, Matt; Ellis, Ronald J.

    2015-01-01

    Background Measurement of HIV DNA-bearing cells in cerebrospinal fluid (CSF) is challenging because few cells are present. We present a novel application of the sensitive droplet digital (dd)PCR in this context. Methods We analyzed CSF cell pellets and paired peripheral blood mononuclear cells (PBMC) from 28 subjects, 19 of whom had undetectable HIV RNA (<48 copies/mL) in both compartments. We extracted DNA from PBMC using silica-based columns and used direct lysis on CSF cells. HIV DNA and the host housekeeping gene (RPP30) were measured in CSF and PBMC by (dd)PCR. We compared HIV DNA levels in virally-suppressed and-unsuppressed subgroups and calculated correlations between HIV DNA and RNA levels in both compartments using non-parametric tests. Results HIV DNA was detected in 18/28 (64%) CSF cell pellets, including 10/19 (53%) samples with undetectable HIV RNA. HIV DNA levels in CSF cell pellets were not correlated with RPP30 (p = 0.3), but correlated positively with HIV RNA in CSF (p = 0.04) and HIV DNA in PBMC (p = 0.03). Cellular HIV DNA in CSF was detected in comparable levels in HIV RNA-suppressed and unsuppressed subjects (p = 0.14). In contrast, HIV DNA levels in PBMC were significantly lower in HIV RNA-suppressed than in unsuppressed subjects (p = 0.014). Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001). Conclusions Despite low mononuclear cell numbers in CSF, HIV DNA was detected in most virally suppressed individuals. In contrast to PBMC, suppressive ART was not associated with lower HIV DNA levels in CSF cells, compared to no ART, perhaps due to poorer ART penetration, slower decay of HIV DNA, or enrichment of HIV DNA-bearing mononuclear cells into the CSF, compared to blood. Future studies should determine what fraction of HIV DNA is replication-competent in CSF leukocytes, compared to PBMC. PMID:26431315

  2. Stability of carbon nanotube yarn biofuel cell in human body fluid

    NASA Astrophysics Data System (ADS)

    Kwon, Cheong Hoon; Lee, Jae Ah; Choi, Young-Bong; Kim, Hyug-Han; Spinks, Geoffrey M.; Lima, Márcio D.; Baughman, Ray H.; Kim, Seon Jeong

    2015-07-01

    High performance with stability, easy-handling electrodes, and biofluid-flow controllable system with mechanical strength of the biofuel cell can be considered as the critical issues for future human body implant. These three challenges are sufficiently considered by using the effective platform regarding the high surface area from multi-walled carbon nanotube-conducting polymer with poly(3,4-ethylenedioxythiophene), and size/shape dependent flexible yarn electrodes for the implantation of biofuel cell. High power biofuel cell of mW cm-2 range in physiological condition (low glucose-containing phosphate buffered saline solution and human blood serum) controlling the stirring degree is also first demonstrated for future implantation in this study. Biofuel cells for future implantation in human body vitally require long-term stability and high power outputs. We have demonstrated that a high-surface area yarn-based biofuel cell retained over 70% of its initial power output after an extended 20 days period of continuous operation in human blood serum, while delivering a power density of ∼1.0 mW cm-2. Subsequently, our enhanced enzymatic biofuel cell system would be potentially used as an innovative power source for the next generation implantable electronics.

  3. Gene expression of single human mesenchymal stem cell in response to fluid shear

    PubMed Central

    Zhang, Hu; Kay, Alasdair; Forsyth, Nicholas R; Liu, Kuo-Kang

    2012-01-01

    Stem cell therapy may rely on delivery and homing through the vascular system to reach the target tissue. An optical tweezer model has been employed to exert different levels of shear stress on a single non-adherent human bone marrow–derived mesenchymal stem cell to simulate physiological flow conditions. A single-cell quantitative polymerase chain reaction analysis showed that collagen type 1, alpha 2 (COL1A2), heat shock 70-kDa protein 1A (HSPA1A) and osteopontin (OPN) are expressed to a detectable level in most of the cells. After exposure to varying levels of shear stress, there were significant variations in gene transcription levels across human mesenchymal stem cells derived from four individual donors. Significant trend towards upregulation of COL1A2 and OPN gene expression following shear was observed in some donors with corresponding variations in HSPA1A gene expression. The results indicate that shear stress associated with vascular flow may have the potential to significantly direct non-adherent stem cell expression towards osteogenic phenotypic expression. However, our results demonstrate that these results are influenced by the selection process and donor variability. PMID:22798982

  4. Cerebrospinal fluid.

    PubMed

    Jerrard, D A; Hanna, J R; Schindelheim, G L

    2001-08-01

    A quick and accurate diagnosis of maladies affecting the central nervous system (CNS) is imperative. Procurement and analysis of cerebrospinal fluid (CSF) are paramount in helping the clinician determine a patient's clinical condition. Various staining methods, measurement of white blood cell counts, glucose and protein levels, recognition of xanthochromia, and microbiologic studies are CSF parameters that are collectively important in the ultimate determination by a clinician of the presence or absence of a catastrophic CNS condition. Many of these CNS parameters have significant limitations that should be recognized to minimize under treating patients with catastrophic illness. PMID:11489408

  5. Numerical simulation of osteocyte cell in response to directional mechanical loadings and mechanotransduction analysis: Considering lacunar-canalicular interstitial fluid flow.

    PubMed

    Joukar, Amin; Niroomand-Oscuii, Hanieh; Ghalichi, Farzan

    2016-09-01

    The osteocyte cell is a bone cell that also functions as a bone mechanosensor. In this work, a three-dimensional (3D) fluid-structure interaction (FSI) model of an osteocyte cell under different mechanical loading conditions was used to obtain a better understanding of osteocyte cell behavior under different physiological conditions. In the current study, both fluid and solid parts of osteocyte cell were considered in order to allow for more accurate results. Five different loading conditions have been applied to the osteocyte cell, and consequently the different interstitial fluid flow velocities and shear stresses have been investigated. Furthermore, using a mathematical model, the change in the stimulus function value with shear stress and NO enzyme was revealed. This work suggests that changes in osteocyte morphology and direction of loadings affect cell stimulation. It was found that cell is mostly stimulated and expanded in the direction experiencing the most shear stress. Finally, the amount of cell stimulation was shown quantitatively and there was strong dependency between stimulus function, shear stress, calcium, and NO concentration. PMID:27393805

  6. The importance of correcting for variable probe-sample interactions in AFM-IR spectroscopy: AFM-IR of dried bacteria on a polyurethane film.

    PubMed

    Barlow, Daniel E; Biffinger, Justin C; Cockrell-Zugell, Allison L; Lo, Michael; Kjoller, Kevin; Cook, Debra; Lee, Woo Kyung; Pehrsson, Pehr E; Crookes-Goodson, Wendy J; Hung, Chia-Suei; Nadeau, Lloyd J; Russell, John N

    2016-08-01

    AFM-IR is a combined atomic force microscopy-infrared spectroscopy method that shows promise for nanoscale chemical characterization of biological-materials interactions. In an effort to apply this method to quantitatively probe mechanisms of microbiologically induced polyurethane degradation, we have investigated monolayer clusters of ∼200 nm thick Pseudomonas protegens Pf-5 bacteria (Pf) on a 300 nm thick polyether-polyurethane (PU) film. Here, the impact of the different biological and polymer mechanical properties on the thermomechanical AFM-IR detection mechanism was first assessed without the additional complication of polymer degradation. AFM-IR spectra of Pf and PU were compared with FTIR and showed good agreement. Local AFM-IR spectra of Pf on PU (Pf-PU) exhibited bands from both constituents, showing that AFM-IR is sensitive to chemical composition both at and below the surface. One distinct difference in local AFM-IR spectra on Pf-PU was an anomalous ∼4× increase in IR peak intensities for the probe in contact with Pf versus PU. This was attributed to differences in probe-sample interactions. In particular, significantly higher cantilever damping was observed for probe contact with PU, with a ∼10× smaller Q factor. AFM-IR chemical mapping at single wavelengths was also affected. We demonstrate ratioing of mapping data for chemical analysis as a simple method to cancel the extreme effects of the variable probe-sample interactions. PMID:27403761

  7. Elastic modulus, oxidation depth and adhesion force of surface modified polystyrene studied by AFM and XPS

    NASA Astrophysics Data System (ADS)

    Lubarsky, G. V.; Davidson, M. R.; Bradley, R. H.

    2004-06-01

    AFM and XPS have been used to investigate the surface and near-surface properties of polystyrene (PS) substrates which have been subjected to one of three controlled surface modification processes performed in situ in a specially constructed cell. The cell was fitted to a Digital Instruments Nanoscope III AFM measuring head and allowed close control of the gaseous environment and made it possible to UV irradiate the sample during AFM measurements. Treatments were carried out using UV at 184.9 and 253.7 nm wavelengths, in oxygen (UV-ozone), and in nitrogen (UV-only). Polystyrene surfaces were also modified by an exposure to an atmosphere of ozone in the absence of UV (ozone-only). Data show that adhesion force is highest between tip and sample for the UV-ozone exposed surfaces and that the adhesion force increases with sample exposure time. Exposure to UV-only or ozone alone results in lower ultimate adhesion levels with a slower rate of increase with exposure time. Evaluation of Young's modulus for unmodified PS gave a value of 3.37 (±0.52) GPa which agrees well with the textbook value which ranges from 2 to 4 GPa depending on the measurement technique. A 60 s exposure to combined UV-ozone resulted in the formation of a surface layer with a modulus at the surface of 1.25 (±0.19) GPa which increased to 2.5 (±0.37) GPa at a depth of 3.5 nm. The sample exposed for 60 s to UV-only had a Young's modulus of 2.6 (±0.39) GPa but showed no reduced modulus layer at the surface. The modulus of the ozone-only treated material was the least affected with a decrease of around 0.75 GPa with some evidence for a surface layer with a modulus ranging from 2.6 (±0.39) GPa at the surface to 3.2 (±0.48) GPa at a depth of 2 nm. XPS analyses reveal that the oxygen content of the modified surfaces decreased in the order of UV-ozone > UV > ozone with approximate concentrations for a 60 s exposure of 5, 0.7 and 0.05 at.%, respectively. Friction force imaging of patterned surfaces

  8. AFM CHARACTERIZATION OF RAMAN LASER INDUCED DAMAGE ON CDZNTECRYSTAL SURFACES

    SciTech Connect

    Teague, L.; Duff, M.

    2008-10-07

    High quality CdZnTe (or CZT) crystals have the potential for use in room temperature gamma-ray and X-ray spectrometers. Over the last decade, the methods for growing high quality CZT have improved the quality of the produced crystals however there are material features that can influence the performance of these materials as radiation detectors. The presence of structural heterogeneities within the crystals, such as twinning, pipes, grain boundaries (polycrystallinity), and secondary phases (SPs) can have an impact on the detector performance. There is considerable need for reliable and reproducible characterization methods for the measurement of crystal quality. With improvements in material characterization and synthesis, these crystals may become suitable for widespread use in gamma radiation detection. Characterization techniques currently utilized to test for quality and/or to predict performance of the crystal as a gamma-ray detector include infrared (IR) transmission imaging, synchrotron X-ray topography, photoluminescence spectroscopy, transmission electron microscopy (TEM), atomic force microscopy (AFM) and Raman spectroscopy. In some cases, damage caused by characterization methods can have deleterious